Complex Kinetic Behavior Research Articles

Activation of Coq6p, a FAD Monooxygenase Involved in Coenzyme Q Biosynthesis, by Adrenodoxin Reductase/Ferredoxin.

Adrenodoxin reductase (AdxR) plays a pivotal role in electron transfer, shuttling electrons between NADPH and iron/sulfur adrenodoxin proteins in mitochondria. This electron transport system is essential for P450 enzymes involved in various endogenous biomolecules biosynthesis. Here, we present an in-depth examination of the kinetics governing the reduction of human AdxR by NADH or NADPH. Our results highlight the efficiency of human AdxR when utilizing NADPH as a flavin reducing agent. Nevertheless, akin to related flavoenzymes such as cytochrome P450 reductase, we observe that low NADPH concentrations hinder flavin reduction due to intricate equilibrium reactions between the enzyme and its substrate/product. Remarkably, the presence of MgCl2 suppresses this complex kinetic behavior by decreasing NADPH binding to oxidized AdxR, effectively transforming AdxR into a classical Michaelis-Menten enzyme. We propose that the addition of MgCl2 may be adapted for studying the reductive half-reactions of other flavoenzymes with NADPH. Furthermore, in vitro experiments provide evidence that the reduction of the yeast flavin monooxygenase Coq6p relies on an electron transfer chain comprising NADPH-AdxR-Yah1p-Coq6p, where Yah1p shuttles electrons between AdxR and Coq6p. This discovery explains the previous in vivo observation that Yah1p and the AdxR homolog, Arh1p, are required for the biosynthesis of coenzyme Q in yeast.

A physiologically-based kinetic (PBK) model for work-related diisocyanate exposure: Relevance for the design and reporting of biomonitoring studies

Diisocyanates are highly reactive substances and known causes of occupational asthma. Exposure occurs mainly in the occupational setting and can be assessed through biomonitoring which accounts for inhalation and dermal exposure and potential effects of protective equipment. However the interpretation of biomonitoring data can be challenging for chemicals with complex kinetic behavior and multiple exposure routes, as is the case for diisocyanates. To better understand the relation between external exposure and urinary concentrations of metabolites of diisocyanates, we developed a physiologically based kinetic (PBK) model for methylene bisphenyl isocyanate (MDI) and toluene di-isocyanate (TDI). The PBK model covers both inhalation and dermal exposure, and can be used to estimate biomarker levels after either single or chronic exposures. Key parameters such as absorption and elimination rates of diisocyanates were based on results from human controlled exposure studies. A global sensitivity analysis was performed on model predictions after assigning distributions reflecting a mixture of parameter uncertainty and population variability.Although model-based predictions of urinary concentrations of the degradation products of MDI and TDI for longer-term exposure scenarios compared relatively well to empirical results for a limited set of biomonitoring studies in the peer-reviewed literature, validation of model predictions was difficult because of the many uncertainties regarding the precise exposure scenarios that were used. Sensitivity analyses indicated that parameters with a relatively large impact on model estimates included the fraction of diisocyanates absorbed and the binding rate of diisocyanates to albumin relative to other macro molecules.We additionally investigated the effects of timing of exposure and intermittent urination, and found that both had a considerable impact on estimated urinary biomarker levels. This suggests that these factors should be taken into account when interpreting biomonitoring data and included in the standard reporting of isocyanate biomonitoring studies.

Silver-ruthenium bimetallic nanoparticles as sacrificial heterogeneous persulfate activator in situ chemical oxidation of dye

Silver-ruthenium bimetallic (Ag-Ru) synthesized using green chemical co-reduction method. The valence state, composition, crystallinity, shape, and size of Ag-Ru were evaluated by using XPS, EDX, XRD, SEM, and TEM measurements. The XPS analysis indicates that charge transfer takes place contacting silver and ruthenium moieties. To present the advantages of Ag-Ru NPs as heterogeneous activator, sulfate radical based advanced oxidation processes for patent blue V dye degradation was chosen as a model contaminant. The Ag-Ru/S2O82− system shows great catalytic activity at lower dye concentration, pH, increasing the dosage of NPs, and persulfate. The activated system showed a much higher dye oxidation rate compared to either Ag-Ru or S2O82− alone. The degradation percentage decreases with increasing the dye concentration as well as pH of the reaction media. The persulfate exhibits complex kinetic behavior on the degradation of dye. The activation energy for the system was 57.7 kJ/mol calculated from Arrhenius equation. The oxidation of dye follows pseudo-first order kinetics (kapp = 0.029 min−1). The excellent activation performance of Ag-Ru might be due to the synergistic effect of silver and ruthenium. The reaction mechanism of Ag-Ru bimetallic activation of persulfate was elucidated and discussed. The electron transfer from Ru3+ / Ru2+ and Ag2+/Ag+ plays significant role in the regeneration of active radicals. In addition, Ag-Ru was under goes complete oxidative dissolution with persulfate.

Using Synchrotron Techniques, Investigation of Electrochemical Interfaces in Ni-Rich NMC and Sulfide Electrolytes in All-Solid-State Lithium Metal Batteries

a Nano-electrochemistry Laboratory, Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei 106, Taiwan, R.O.C b National Synchrotron Radiation Research Center (NSRRC), Hsinchu, 30076, Taiwan, R.O.CIn all-solid-state lithium metal batteries enable long cyclability of high voltage oxides cathode persistent problem for the large scale application as their underprivileged interfacial steadiness in contrast to sulfide solid-state electrolyte. In this context, the interfaces of the solid electrolyte and Ni-rich NMC811 active material are looked upon as interfacial chemical responses induced by delithiation. In this study, we monitor the impedance progress at the unstable electrode|electrolyte interface due to the electrochemical interfacial response and help us understand the complex nature of reactivity and degradation kinetics with the solid-solid interface redox decomposition, which makes decoupling each effect difficult. we investigated the interfacial phenomenon between LPSC and high voltage cathode NMC811. The effects of spontaneous retort by the side of the interface were separated, and the intrinsic electrochemical decomposition of LPSC was quantified. Moreover, we show that the notch of interfacial degradation surges and the presence of oxidation mechanisms. At the higher delithiation stage, the cathode might twitch structural defenselessness and oxygen utter and resulting in further stark degradation. This complex kinetic degradation behavior was investigated at the solid-solid interface in a delithiation NMC811 and SSE based on the local oxidation state of NMC811, and LPSC SE interfacial chemical response. In this work, we used various characterization techniques to investigate the interfacial phenomenon between LPSC|NMC811 combining EIS and advanced synchrotron techniques such as sXAS, XPS, XRF-XANES mapping, and In-situ Raman spectroscopy. Keywords: delithiation, Ni-rich cathode, Sulfide-solid-state electrolyte, interfacial reaction, Synchrotron XPS, XRF.

Reaction kinetics of OH radicals with 1,3,5-trimethyl benzene: An experimental and theoretical study

Alkylated aromatics are ubiquitous in transportation fuels. 1,3,5-Trimethyl benzene (135TMB) is a popular surrogate for the aromatic content of distillate fuels due to its symmetry (point group, C3h), which facilitates the construction of an accurate chemical kinetic model. The reaction of OH radicals with 135TMB plays a crucial role in the oxidation kinetics of 135TMB. In this work, the reaction kinetics of OH-initiated oxidation of 135TMB were investigated behind reflected shock waves over 975–1318 K and atmospheric pressure. The reaction was followed by monitoring OH radicals near 307 nm. The rate coefficients were extracted from detailed chemical kinetic modeling of OH concentration-time profiles. Our measured data clearly showed a positive temperature dependence, in contrast to the negative temperature dependence of the literature low-temperature data. At 1000 K, our measured rate coefficient is 1.3 × 10−11 cm3 molecule−1 s−1, which is roughly a factor of 5 lower than the room-temperature data reported in the literature. This observation reflects the complex nature of the OH + 135TMB reaction, similar to that observed for various aromatics + OH chemical systems. Our measurements did not show any discernible pressure dependence over the narrow pressure range of 870 – 1148 Torr. The title reaction has several possible channels in the reactive potential energy surface. The importance of each channel was characterized using ab initio/RRKM-ME calculations over T = 200–2000 K and P = 0.76 -7600 Torr. Our analyses revealed that addition channels and hydrogen abstraction from the methyl site have negative energy barriers. The reaction was found to undergo almost exclusively (∼93%) via the addition channel under ambient conditions. However, beyond 600 K, the abstraction channels take the lead, yielding the positive T-dependence of the overall rate coefficient. Although addition channels display a sharp fall-off behavior beyond 500 K, the general rate coefficients are pressure-independent. The title reaction shows a complex kinetic behavior due to competing channels whose contribution changes significantly with temperature. Our theoretical calculations nicely reproduced the complex T-dependence of the reaction. After adjusting the barrier height, our theory remarkably captured the positive T-dependence of our high-T kinetic data and the negative T-dependence of the low-T literature data. To our knowledge, this is the first detailed study on the reaction kinetics of 135TMB with OH radicals. The reported rate data will be helpful for the combustion modeling of alkylated aromatic species.

Exploring the reactivity of aliphatic and phenolic hydroxyl groups in lignin hydrogenolysis oil towards urethane bond formation

Lignin is the second most abundant natural polymer that exhibits a complex structure with various amounts of aliphatic and phenolic hydroxides. The use of lignin as a source of polyol to make lignin-based polyurethanes (PUs) has been the subject of intense studies in recent years. It is well known that there is a difference in reactivity between aliphatic and aromatic hydroxyl groups towards isocyanates. For simple model systems using conventional fossil-fuel based polyols the overall kinetics are well known. However, unveiling the complex kinetic behavior obtained when combining an isocyanate with lignin derived components exhibiting different types of hydroxyl groups is challenging. Therefore, an in-depth investigation using lignin model compounds and lignin hydrogenolysis oil to elucidate these differences of reactivity is presented and will be invaluable towards the development of lignin-based PUs. Understanding the kinetics involved will lead to optimized synthetic protocols and a higher valorization potential for lignin-based PUs. In this work, various monolignol model compounds are studied concerning their reactivity to aliphatic and aromatic isocyanates. Isothermal experiments revealed the kinetic rate constants and reaction orders while non-isothermal DSC was used to obtain the activation energies through the Friedman isoconversion model-free-kinetics (MFK) approach. These results were confirmed with FT-IR and 1H NMR. It was found that in all cases, aliphatic hydroxide groups react the fastest with aromatic isocyanates while aromatic hydroxide groups react the slowest with aromatic isocyanates. Reactions with aliphatic isocyanates, independent of the hydroxyl type, fall in between these extremes shown by the aromatic isocyanates. Apparent activation energies ranging from 55.2 kJ·mol-1 to 39.5 kJ·mol-1 were found with corresponding reaction rate constants ranging from 5.19·10-3 M1−n s-1 to 1.04·10-3 M1−n s-1. The presented work will be a capstone for the further valorization of lignin as a material for renewable PUs.

Coverage Fluctuations and Correlations in Nanoparticle-Catalyzed Diffusion-Influenced Bimolecular Reactions

The kinetic processes in nanoparticle-based catalysis are dominated by large fluctuations and spatiotemporal heterogeneities, in particular for diffusion-influenced reactions which are far from equilibrium. Here, we report results from particle-resolved reaction-diffusion simulations of steady-state bimolecular reactions catalyzed on the surface of a single, perfectly spherical nanoparticle. We study various reactant adsorption and diffusion regimes, in particular considering the crowding effects of the reaction products. Our simulations reveal that fluctuations, significant coverage cross-correlations, transient self-poisoning, related domain formation, and excluded-volume effects on the nanoparticle surface lead to a complex kinetic behavior, sensitively tuned by the balance between adsorption affinity, mixed 2D and 3D diffusion, and chemical reaction propensity. The adsorbed products are found to influence the correlations and fluctuations, depending on overall reaction speed, thereby going beyond conventional steric (e.g., Langmuir-like) product inhibition mechanisms. We summarize our findings in a state diagram depicting the nonlinear kinetic regimes by an apparent surface reaction order in dependence of the intrinsic reaction propensity and adsorption strength. Our study using a simple, perfectly spherical, and inert nanocatalyst demonstrates that spatiotemporal heterogeneities are intrinsic to the reaction-diffusion problem and not necessarily caused by any dynamical surface effects from the catalyst (e.g., dynamical surface reconstruction), as often argued.

Reduced-order model for CaO carbonation kinetics measured using micro-fluidized bed thermogravimetric analysis

A reduced-order model was developed to describe the CaO carbonation kinetics by simplifying the detailed one-dimensional rate-equation-based grain model. The reduced-order model was derived using the Thiele modulus method and is an analytical model, which can be easily used in the reactor scale model or computational fluid dynamics (CFD) simulation. In the reduced-order model, physical/chemical steps occurring during the carbonation process were considered, including external diffusion of gas around the particle, intraparticle diffusion, surface reaction, product islands growth and product layer diffusion. This model can be used to investigate the complex kinetic behaviors such as reaction controlled steps, kinetics transition from chemical reaction to product layer diffusion, and the effect of particle structure change on intraparticle diffusion, such as pore plugging phenomenon. The prediction accuracy of reduced-order model was verified by the detailed one-dimensional model. In order to demonstrate the application of the reduced-order model for reactor scale analysis, the CaO carbonation kinetics under different temperatures (600–750 °C), CO2 concentrations (25–75 vol%) and particle sizes (0.2–3 mm) were measured using a novel micro-fluidized bed thermogravimetric analysis (MFB-TGA) method, which is based on real-time mass measurement of CaO sample in a fluidizing state. The reduced-order model was implemented into a K-L two-phase bubbling bed reactor model to analyze the experimental data of a micro-fluidized bed reactor. Results indicated that the two-stage behavior of carbonation kinetics can be described successfully using the model, and the effect of particle structure change on pore plugging and intraparticle diffusion can be well predicted and explained. The carbonation is first-order reaction when the CO2 partial pressure is within 75% (1 bar). The effect of particle size on carbonation kinetics shows three-zone characteristics of reaction/diffusion combined controlling mechanism, and was discussed in detail. The reduced-order model provides a promising application for describing multiscale mechanisms (surface, particle and reactor) of carbonation kinetics.

Structural and Chemical Biology of the Interaction of Cyclooxygenase with Substrates and Non-Steroidal Anti-Inflammatory Drugs.

Cyclooxgenases are key enzymes of lipid signaling. They carry out the first step in the production of prostaglandins, important mediators of inflammation, pain, cardiovascular disease, and cancer, and they are the molecular targets for nonsteroidal anti-inflammatory drugs, which are among the oldest and most chemically diverse set of drugs known. Homodimeric proteins that behave as allosterically modulated, functional heterodimers, the cyclooxygenases exhibit complex kinetic behavior, requiring peroxide-dependent activation and undergoing suicide inactivation. Due to their important physiological and pathophysiological roles and keen interest on the part of the pharmaceutical industry, the cyclooxygenases have been the focus of a vast array of structural studies, leading to the publication of over 80 crystal structures of the enzymes in complex with substrates or inhibitors supported by a wealth of functional data generated by site-directed mutation experiments. In this review, we explore the chemical biology of the cyclooxygenases through the lens of this wealth of structural and functional information. We identify key structural features of the cyclooxygenases, break down their active site into regional binding pockets to facilitate comparisons between structures, and explore similarities and differences in the binding modes of the wide variety of ligands (both substrates and inhibitors) that have been characterized in complex with the enzymes. Throughout, we correlate structure with function whenever possible. Finally, we summarize what can and cannot be learned from the currently available structural data and discuss the critical intriguing questions that remain despite the wealth of information that has been amassed in this field.

Real-Time Quantitative In-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human Cells Using Multivariate Curve Resolution.

In-cell NMR can investigate protein conformational changes at atomic resolution, such as those changes induced by drug binding or chemical modifications, directly in living human cells, and therefore has great potential in the context of drug development as it can provide an early assessment of drug potency. NMR bioreactors can greatly improve the cell sample stability over time and, more importantly, allow for recording in-cell NMR data in real time to monitor the evolution of intracellular processes, thus providing unique insights into the kinetics of drug-target interactions. However, current implementations are limited by low cell viability at >24 h times, the reduced sensitivity compared to “static” experiments and the lack of protocols for automated and quantitative analysis of large amounts of data. Here, we report an improved bioreactor design which maintains human cells alive and metabolically active for up to 72 h, and a semiautomated workflow for quantitative analysis of real-time in-cell NMR data relying on Multivariate Curve Resolution. We apply this setup to monitor protein–ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing further analysis by fitting with kinetic models. This unique approach can therefore be applied to investigate complex kinetic behaviors of macromolecules in a cellular setting, and could be extended in principle to any real-time NMR application in live cells.

Dynamic evolution of pore structures of hydrate-bearing sediments induced by step-wise depressurization

Pore structures are of great importance to study gas production from natural gas hydrate reservoir. The dynamic evolution of pore structures over the course of methane hydrate dissociation and their association with particle size distribution of hydrate-bearing sediments remain poorly understood. In this study, a combination of X-ray computed tomography (CT) observation and pore network modeling was applied to exploring the dynamic evolution of pore structures with hydrate dissociation induced by depressurization in two initially brine-unsaturated sediments with different particle size distributions. Methane hydrates were synthesized in two silica sands, each of which is with different particle size distributions, at 2.0°C and 8.5 MPa. The reaction process continued nearly three weeks until the pore pressure and temperature inside the reaction vessel did not have significant changes for at least 7 d, to provide sufficient time for methane hydrate in sediment pores to reach steady spatial distribution. The X-ray micro-focus CT observation of methane hydrate dissociation, which was induced by step-wise depressurization, was further performed, to obtain a series of high-resolution CT images of hydrate-bearing sediments at different degrees of hydrate saturation. The corresponding pore structures of the two sediments with different hydrate saturation are studied by the digital image processing methods, including image filter, 2-D threshold segmentation of binarized CT images, 3-D digital core reconstruction, and topological extraction of actual pore structure. To overcome the obstacle caused by the similar X-ray attenuation coefficients of methane hydrate and deionized water, the phase-contrast between methane hydrate and pore water was enhanced by the addition of NaCl into pore water. To have a better configuration about the distributions of materials in sediments, the bulky patchy-like hydrate adhering to the inner wall of the reaction vessel or occupying macroscopic pores was used to calibrate the CT values of sand, water, gas, and methane hydrate in pore space. Based on the above results, the dynamic evolution of sediment pore structures with hydrate dissociation, which was induced by step-wise depressurization, and their association with particle size distribution were investigated. The results indicate that the morphology of gas hydrate in sediments is largely heterogeneous, and the dissociation of methane hydrate generally starts from the contacting place between gas and hydrate. In the early stage of hydrate dissociation, several types of hydrate morphology appear, including pore-filling and grain-cementing, while in the late stage of hydrate dissociation, grain-cementing hydrate is dominant. As methane hydrate dissociates, the average pore throat radius, porosity, absolute permeability and two-phase percolation zone in the sediments will gradually increase, but the coordination number, shape factor, and irreducible water saturation will change in an opposite trend. Due to the complex kinetic behavior of hydrate dissociation, the average pore radius usually gradually decreases in the early stage of hydrate dissociation, followed by a sharp increase when the hydrate saturation is below a critical value of

Association of Model Neurotransmitters with Lipid Bilayer Membranes

Aimed at reproducing the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neurotransmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflection measurements on artificial membranes, so-called sparsely tethered bilayer lipid membranes, reveal the structural aspects of neurotransmitters’ association with zwitterionic and anionic bilayers. We thus establish that serotonin interacts nonspecifically with the membrane at physiologically relevant concentrations, whereas γ-aminobutyric acid does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity, and neutron reflectometry shows that it penetrates the membrane deeply, whereas γ-aminobutyric is excluded from the bilayer.

Kinetics of iron(III)-catalyzed oxidation of arsenic(III) in acidic solutions with SO2/O2 gas mixture using different iron sources

Oxidation of arsenic(III) to arsenic(V) is very important in arsenic removal and immobilization operations. Limited studies on arsenic(III) oxidation at pH below 1.5 as is the case of acid plant and leaching process effluents have been reported. In this work, the kinetics of As(III) oxidation with SO2/O2 gas mixture at low pH (0.3–1.0) sulfuric acid solutions was investigated in the presence of different iron sources as catalyst: (i) Fe(III); (ii) Fe(II); and (iii) FeOOH residue. In the absence of Fe(III) the initial rate of As(III) oxidation at 90 °C is 0.6 mmol/L·min increasing to 1.1 mmol/L·min with Fe(III)/As(III) molar ratio = 0.1. Higher additions of Fe(III) bring only modest increase in rate. In the presence of Fe(II) the oxidation of As(III) is delayed initially due to competing oxidation of Fe(II) subsequently accelerating with the build-up of Fe(III), which acts as catalyst. Use of FeOOH (industrial goethite residue) provides a cost-effective source of Fe(III) catalytic species while simultaneously acts as neutralizing agent. A complex kinetic behavior is followed. Oxidation is reaction-controlled over the temperature range 25–50 °C shifting to mixed regime control from 70 to 80 °C becoming finally fully mass-transfer controlled at 90 °C. The catalytic role of Fe(III) is rationalized on the basis of a radical chain mechanism.

Bayesian-Estimated Hierarchical HMMs Enable Robust Analysis of Single-Molecule Kinetic Heterogeneity

Single-molecule kinetic experiments allow the reaction trajectories of individual biomolecules to be directly observed, eliminating the effects of population averaging and providing a powerful approach for elucidating the kinetic mechanisms of biomolecular processes. A major challenge to the analysis and interpretation of these experiments, however, is the kinetic heterogeneity that almost universally complicates the recorded single-molecule signal versus time trajectories (i.e., signal trajectories). Such heterogeneity manifests as changes and/or differences in the transition rates that are observed within individual signal trajectories or across a population of signal trajectories. Because characterizing kinetic heterogeneity can provide critical mechanistic information, we have developed a computational method that effectively and comprehensively enables such analysis. To this end, we have developed a computational algorithm and software program, hFRET, that uses the variational approximation for Bayesian inference to estimate the parameters of a hierarchical hidden Markov model, thereby enabling robust identification and characterization of kinetic heterogeneity. Using simulated signal trajectories, we demonstrate the ability of hFRET to accurately and precisely characterize kinetic heterogeneity. In addition, we use hFRET to analyze experimentally recorded signal trajectories reporting on the conformational dynamics of ribosomal pre-translocation (PRE) complexes. The results of our analyses demonstrate that PRE complexes exhibit kinetic heterogeneity, reveal the physical origins of this heterogeneity, and allow us to expand the current model of PRE complex dynamics. The methods described here can be applied to signal trajectories generated using any type of signal and can be easily extended to the analysis of signal trajectories exhibiting more complex kinetic behaviors. Moreover, variations of our approach can be easily developed to integrate kinetic data obtained from different experimental constructs and/or from molecular dynamics simulations of a biomolecule of interest.

Noncompetitive antagonists induce cooperative AMPA receptor channel gating.

Glutamate is released from presynaptic nerve terminals in the central nervous system (CNS) and spreads excitation by binding to and activating postsynaptic iGluRs. Of the potential glutamate targets, tetrameric AMPA receptors mediate fast, transient CNS signaling. Each of the four AMPA subunits in the receptor channel complex is capable of binding glutamate at its ligand-binding domains and transmitting the energy of activation to the pore domain. Homotetrameric AMPA receptor channels open in a stepwise manner, consistent with independent activation of individual subunits, and they exhibit complex kinetic behavior that manifests as temporal shifts between four different conductance levels. Here, we investigate how two AMPA receptor-selective noncompetitive antagonists, GYKI-52466 and GYKI-53655, disrupt the intrinsic step-like gating patterns of maximally activated homotetrameric GluA3 receptors using single-channel recordings from cell-attached patches. Interactions of these 2,3-benzodiazepines with residues in the boundary between the extracellular linkers and transmembrane helical domains reorganize the gating behavior of channels. Low concentrations of modulators stabilize open and closed states to different degrees and coordinate the activation of subunits so that channels open directly from closed to higher conductance levels. Using kinetic and structural models, we provide insight into how the altered gating patterns might arise from molecular contacts within the extracellular linker-channel boundary. Our results suggest that this region may be a tunable locus for AMPA receptor channel gating.

Amplified detection of single base mismatches with the competing-strand assay reveals complex kinetic and thermodynamic behavior of strand displacement at the electrode surface

Detection of single-base mismatches with respect to a probe strand has been a predominant pursuit in electrochemical biosensor efforts, due to links found between single nucleotide polymorphisms (SNPs) and the predisposition to various diseases. We report an osmium tetroxide bipyridine-based, thermally-controlled, competitive-strand electrochemical assay to allow amplified detection of single-base mismatches. Optimally designed competitive-strand displacement and hybridization temperature allows us to distinguish the single-mismatched-target from the fully complementary sequence with unambiguous, highly reproducible, robust signal differences of over 90%. Furthermore, we find a complex interplay between the position of the redox label, variations in strand displacement kinetics due to mismatches incorporated into the competitive strand, and alterations in the melting temperature of DNA duplexes tethered on the gold surface, when probed by square-wave voltammetry. These insights will apply to any surface-tethered DNA-based electrochemical biosensor, and can help with understanding complex phenomena involved in these types of assays.

Replication of Simulated Prebiotic Amphiphilic Vesicles in a Finite Environment Exhibits Complex Behavior That Includes High Progeny Variability and Competition.

We studied the simulated replication and growth of prebiotic vesicles composed of 140 phospholipids and cholesterol using our R-GARD (Real Graded Autocatalysis Replication Domain) formalism that utilizes currently extant lipids that have known rate constants of lipid-vesicle interactions from published experimental data. R-GARD normally modifies kinetic parameters of lipid-vesicle interactions based on vesicle composition and properties. Our original R-GARD model tracked the growth and division of one vesicle at a time in an environment with unlimited lipids at a constant concentration. We explore here a modified model where vesicles compete for a finite supply of lipids. We observed that vesicles exhibit complex behavior including initial fast unrestricted growth, followed by intervesicle competition for diminishing resources, then a second growth burst driven by better-adapted vesicles, and ending with a final steady state. Furthermore, in simulations without kinetic parameter modifications (“invariant kinetics”), the initial replication was an order of magnitude slower, and vesicles' composition variability at the final steady state was much lower. The complex kinetic behavior was not observed either in the previously published R-GARD simulations or in additional simulations presented here with only one lipid component. This demonstrates that both a finite environment (inducing selection) and multiple components (providing variation for selection to act upon) are crucial for portraying evolution-like behavior. Such properties can improve survival in a changing environment by increasing the ability of early protocellular entities to respond to rapid environmental fluctuations likely present during abiogenesis both on Earth and possibly on other planets. This in silico simulation predicts that a relatively simple in vitro chemical system containing only lipid molecules might exhibit properties that are relevant to prebiotic processes. Key Words: Phospholipid vesicles—Prebiotic compartments—Prebiotic vesicle competition—Prebiotic vesicle variability. Astrobiology 18, 419–430.

Kinetics of Membrane Bending by Protein Crowding

Dynamic shape changes in phospholipid membranes are considered critical to a number of important cellular processes. Such structural changes are thought to be driven by a number of possible mechanisms. Recently, the effects of protein crowding on shaping and remodeling membrane surfaces has attracted a lot of interest due to the ubiquity of crowded interfaces in biological systems. While a number of recent studies have confirmed the ability of proteins to drive curvature solely via a colligative, crowding-based mechanism- a number of key fundamental mechanistic details remain unsolved. Specifically, very little is known about the kinetics of this process. What are the timescales of membrane deformation process? How kinetically separated are protein binding and the membrane bending events? Is membrane deformation preceded by a previously unresolved intermediate? To address these questions, we provide a comprehensive study on the kinetics of these events through stopped-flow fluorescence using FRET as the spectroscopic handle. A fluorophore-quencher pair was used to study the protein binding kinetics to the bilayer, while a donor-acceptor pair was used as a reporter for membrane area expansion. We report a complex multi-phasic kinetic behavior for both the protein association and membrane expansion process. The association rates follow a surprising negative trend with respect to increasing protein concentration presumably due to saturation of membrane binding sites owing to protein crowding. We find that the membrane deformation happens at significantly slower timescales relative to the protein binding events. The complexity of the fits suggest that the membrane bending process might be preceded by an unresolved intermediate. Taken together with a predictive theoretical model that is currently underway, we believe that our observations will advance our knowledge about membrane bending driven by protein crowding.

Association of Neurotransmitters with Lipid Bilayer Membranes

Aimed to reproduce the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the standard models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling to neurotransmitters via their membrane interaction. Unlike for anesthetics, experimental verification has been difficult because of the low binding affinities of neurotransmitters to lipid bilayer. We quantify this interaction with surface plasmon resonance (SPR) spectroscopy to measure the equilibrium dissociation constant of neurotransmitter membrane association, as well as on and off rates under flow. Neutron reflectometry (NR) on artificial membranes is used to characterize structural aspects of the association of neurotransmitters with the membranes. Sparsely-tethered bilayer lipid membranes composed of zwitterionic (PC) and anionic (PS and PG) lipids were assembled and their interactions with serotonin, γ-aminobutyric acid (GABA), dopamine, and adenosine studied. SPR shows a wide range of binding affinities for different neurotransmitters. NR shows that the ligand with the largest affinity, serotonin, penetrates the membrane deeply whereas GABA associates with the bilayer peripherally. We establish that some neurotransmitters interact non-specifically with the lipid membrane matrix at physiologically relevant concentration and that this interaction differs vastly for different neurotransmitters.

Cell-Free DNA Kinetics in a Pre-Clinical Model of Head and Neck Cancer

In cancer patients, circulating tumour-derived DNA (ctDNA) levels imperfectly reflect disease burden apparent on medical imaging. Further evaluation of ctDNA levels over time is needed to better understand the correlation with tumour growth and therapeutic response. We describe ctDNA kinetics within an orthotopic, immunocompetent preclinical rabbit model of local-regionally advanced head and neck squamous cell carcinoma (HNSCC). Monitoring primary tumour and metastatic lymph node volume by computed tomography (CT), we observed a correlation between ctDNA levels and tumour burden. We found that ctDNA detection could precede evidence of tumour on CT. Sensitivity and specificity of ctDNA detection in this model was 90.2% (95% C.I.: 76.9–97.3%) and 85.7% (95% C.I.: 67.3–96.0%), respectively. Rapid tumour growth followed by auto-necrosis and tumour volume contraction produced a spike in ctDNA levels, suggesting that viable tumour cells may be required for sustained ctDNA release. Following surgical resection, both ctDNA and total plasma DNA were correlated with recurrent tumour volume. Our results reveal the complex kinetic behaviour of ctDNA and total plasma DNA upon tumour growth or surgery. This pre-clinical model could be useful for future studies focused on elucidating mechanisms of ctDNA release into the circulation from primary and metastatic sites.