Cellular and Acellular Assays for Measuring Oxidative Stress Induced by Ambient and Laboratory-Generated Aerosols.

INTRODUCTION: Many studies have established associations between exposure to air pollution, or atmospheric particulate matter (PM), and adverse health effects. An increasing array of studies have suggested oxidative stress as a possible mechanism by which PM-induced health effects arise, and as a result, many chemical and cellular assays have been developed to study PM-induced oxidant production. Although significant progress has been made in recent years, there are still many gaps in this area of research that have not been addressed. Many prior studies have focused on the aerosol of primary origin (e.g., the aerosol emitted from combustion engines) although the aerosol formed from the oxidation of volatile species, secondary organic aerosol (SOA), has been shown to be the predominant type of aerosol even in urban areas. Current SOA health studies are limited in number, and as such, the health effects of SOA are poorly characterized. Also, there is a lack of perspective in terms of the relative toxicities of different SOA systems. Additionally, although chemical assays have identified some SOA constituents associated with adverse health endpoints, the applicability of these results to cellular responses has not been well established. SPECIFIC AIMS: The overall objective of this study was to better understand the oxidative properties of different types and components of PM mixtures (especially SOA) through systematic laboratory chamber experiments and ambient field studies. The study had four specific aims.1 To develop a cellular assay optimized for measuring reactive oxygen and nitrogen species (ROS/RNS) production resulting from PM exposure and to identify a robust parameter that could represent ROS/RNS levels for comparison with different endpoints.2 To identify ambient PM components associated with ROS/RNS production and evaluate whether results from chemical assays represented cellular responses in terms of ROS/RNS production.3 To investigate and provide perspective on the relative toxicities of SOA formed from common biogenic and anthropogenic precursors under different conditions (e.g., humidity, nitrogen oxides [NOx], and redox-active metals) and identify bulk aerosol properties associated with cellular responses.4 To investigate the effects of photochemical aging on aerosol toxicity. METHODS: Ambient PM samples were collected from urban and rural sites in the greater Atlanta area as part of the Southeastern Center for Air Pollution and Epidemiology (SCAPE) study between June 2012 and October 2013. The concentrations of water-soluble species (e.g., water-soluble organic carbon [WSOC], brown carbon [Br C], and metals) were characterized using a variety of instruments. Samples for this study were chosen to span the observed range of dithiothreitol (DTT) activities.Laboratory studies were conducted in the Georgia Tech Environmental Chamber (GTEC) facility in order to generate SOA under well-controlled photooxidation conditions. Precursors of biogenic origin (isoprene, α-pinene, and ß-caryophyllene) and anthropogenic origin (pentadecane, m-xylene, and naphthalene) were oxidized under various formation conditions (dry vs. humid, NOx, and ammonium sulfate vs. iron sulfate seed particles) to produce SOA of differing chemical composition and mass loading. For the naphthalene system, a series of experiments were conducted with different initial hydrocarbon concentrations to produce aerosols with various degree of oxidation. A suite of instruments was utilized to monitor gas- and particle-phase species. Bulk aerosol properties (e.g., O:C, H:C, and N:C ratios) were measured using a high-resolution time-of-flight aerosol mass spectrometer. Filter samples were collected for chemical oxidative potential and cellular measurements. For the naphthalene system, multiple filter samples were collected over the course of a single experiment to collect aerosols of different photochemical aging.For all filter samples, chemical oxidative potentials were determined for water-soluble extracts using a semiautomated DTT assay system. Murine alveolar macrophages and neonatal rat ventricular myocytes were also exposed to PM samples extracted in cell culture medium to investigate cellular responses. ROS/RNS production was detected using the intracellular ROS/RNS probe, carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFA), whereas cellular metabolic activity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Finally, cytokine production, that is, secreted levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were measured post-exposure using an enzyme-linked immunosorbent assay (ELISA). To identify PM constituents associated with oxidative properties, linear regressions between oxidative properties (cellular responses or DTT activity) and aerosol composition (metals, elemental ratios, etc.) were evaluated using Pearson's correlation coefficient, where the significance was determined using multiple imputation and evaluated using a 95% confidence interval. RESULTS: We optimized several parameters for the ROS/RNS assay, including cell density (2 × 104 cells/well for macrophages and 3.33 × 104 cells/well for cardiomyocytes), probe concentration (10 µM), and sample incubation time (24 hours). Results from both ambient and laboratory-generated aerosols demonstrate that ROS/RNS production was highly dose-dependent and nonlinear with respect to PM dose. Of the dose-response metrics investigated in this study (maximum response, dose at which the response is 10% above the baseline [threshold], dose at which 50% of the response is attained [EC50], rate at which the maximum response is attained [Hill slope], and area under the dose-response curve [AUC]), we found that the AUC was the most robust parameter whose informativeness did not depend on dose range.A positive, significant correlation was observed between ROS/RNS production as represented by AUC and chemical oxidative potential as measured by DTT for ambient samples collected in summer. Conversely, a relatively constant AUC was observed for ambient samples collected in winter regardless of the corresponding DTT activity. We also identified several PM constituents (WSOC, BrC, iron, and titanium) that were significantly correlated with AUC for summer samples. The strong correlation between organic species and ROS/RNS production highlights a need to understand the contribution of organic aerosols to PM-induced health effects. No significant correlations were observed for other ROS/RNS metrics or PM constituents, and no spatial trends were observed.For laboratory-generated aerosol, precursor identity influenced oxidative potentials significantly, with isoprene and naphthalene SOA having the lowest and highest DTT activities, respectively. Both precursor identity and formation condition significantly influenced inflammatory responses induced by SOA exposure, and several response patterns were identified for SOA precursors whose photooxidation products share similar carbon-chain length and functionalities. The presence of iron sulfate seed particles did not have an apparent effect on oxidative potentials; however, a higher level of ROS/RNS production was observed for all SOA formed in the presence of iron sulfate compared with ammonium sulfate. We also identified a significant positive correlation between ROS/RNS production and average carbon oxidation state, a bulk aerosol property. It may therefore be possible to roughly estimate ROS/RNS production using this property, which is readily obtainable. This correlation may have significant implications as aerosols have an atmospheric lifetime of a week, during which average carbon oxidation state increases because of atmospheric photochemical aging. Our results suggest that aerosols might become more toxic as they age in the atmosphere. Finally, in the context of ambient samples, laboratory-generated SOA induced comparable or higher levels of ROS/RNS. Oxidative potentials for all laboratory SOA systems, with the exception of naphthalene (which was higher), were all comparable with oxidative potentials observed in ambient samples.

Equilibrium self-assembly of small RNA viruses.

We propose a description for the quasiequilibrium self-assembly of small, single-stranded (ss) RNA viruses whose capsid proteins (CPs) have flexible, positively charged, disordered tails that associate with the negatively charged RNA genome molecules. We describe the assembly of such viruses as the interplay between two coupled phase-transition-like events: the formation of the protein shell (the capsid) by CPs and the condensation of a large ss viral RNA molecule. Electrostatic repulsion between the CPs competes with attractive hydrophobic interactions and attractive interaction between neutralized RNA segments mediated by the tail groups. An assembly diagram is derived in terms of the strength of attractive interactions between CPs and between CPs and the RNA molecules. It is compared with the results of recent studies of viral assembly. We demonstrate that the conventional theory of self-assembly, which does describe the assembly of empty capsids, is in general not applicable to the self-assembly of RNA-encapsidating virions.

Nonequilibrium statistical mechanics of mixtures of particles in contact with different thermostats.

We introduce a novel type of locally driven systems made of two types of particles (or a polymer with two types of monomers) subject to a chaotic drive with approximately white noise spectrum, but different intensity; in other words, particles of different types are in contact with thermostats at different temperatures. We present complete systematic statistical mechanics treatment starting from first principles. Although we consider only corrections to the dilute limit due to pairwise collisions between particles, meaning we study a nonequilibrium analog of the second virial approximation, we find that the system exhibits a surprisingly rich behavior. In particular, pair correlation function of particles has an unusual quasi-Boltzmann structure governed by an effective temperature distinct from that of any of the two thermostats. We also show that at sufficiently strong drive the uniformly mixed system becomes unstable with respect to steady states consisting of phases enriched with different types of particles. In the second virial approximation, we define nonequilibrium "chemical potentials" whose gradients govern diffusion fluxes and a nonequilibrium "osmotic pressure," which governs the mechanical stability of the interface.

Network Formation by Cross-Hybridization of Complementary Strands to Grafted ssDNA.

When a low density brush of single-stranded DNA (ssDNA) targets end-grafted to a surface is immersed in a solution of complementary ssDNA probes, a regular brush of DNA duplexes is formed by 1:1 hybridization between probe and target DNA. We suggest that in higher density brushes of ssDNA this process competes with cross-hybridization of a target strand to several neighboring probe strands resulting in the formation of a cross-linked DNA network. We analyze a simple 2D model of a dense DNA brush and use analytic methods and computer simulations to find how the conditions for network formation depend on system size and DNA length. We argue that in 3D brushes cross-hybridization will nearly always lead to network formation and suggest that this may explain some intriguing results on dense DNA brushes. Experiments on DNA monolayers and concentrated DNA solutions that could test our predictions are proposed.

Adsorption kinetics of a single polymer on a solid plane.

We study analytically and by means of an off-lattice bead-spring dynamic Monte Carlo simulation model the adsorption kinetics of a single macromolecule on a structureless flat substrate in the regime of strong physisorption. The underlying notion of a "stem-flower" polymer conformation, and the related mechanism of "zipping" during the adsorption process are shown to lead to a Fokker-Planck equation with reflecting boundary conditions for the time-dependent probability distribution function (PDF) of the number of adsorbed monomers. The theoretical treatment predicts that the mean fraction of adsorbed segments grows with time as a power law with a power of (1+nu)-1, where nu approximately 3/5 is the Flory exponent. The instantaneous distribution of train lengths is predicted to follow an exponential relationship. The corresponding PDFs for loops and tails are also derived. The complete solution for the time-dependent PDF of the number of adsorbed monomers is obtained numerically from the set of discrete coupled differential equations and shown to be in perfect agreement with the Monte Carlo simulation results. In addition to homopolymer adsorption, we also study regular multiblock copolymers and random copolymers, and demonstrate that their adsorption kinetics may be considered within the same theoretical model.

How long does it take to pull an ideal polymer into a small hole?

We present scaling estimates for characteristic times taulin and taubr of pulling ideal linear and randomly branched polymers of N monomers into a small hole by a force f. We show that the absorbtion process develops as sequential straightening of folds of the initial polymer configuration. By estimating the typical size of the fold involved into the motion, we arrive at the following predictions, taulin(N) approximately N3/2/f and taubr(N)approximately N5/4/f, and we also confirm them by the molecular dynamics experiment.

How proteins search for their specific sites on DNA: the role of DNA conformation.

It is known since the early days of molecular biology that proteins locate their specific targets on DNA up to two orders-of-magnitude faster than the Smoluchowski three-dimensional diffusion rate. An accepted explanation of this fact is that proteins are nonspecifically adsorbed on DNA, and sliding along DNA provides for the faster one-dimensional search. Surprisingly, the role of DNA conformation was never considered in this context. In this article, we explicitly address the relative role of three-dimensional diffusion and one-dimensional sliding along coiled or globular DNA and the possibility of correlated readsorption of desorbed proteins. We have identified a wealth of new different scaling regimes. We also found the maximal possible acceleration of the reaction due to sliding. We found that the maximum on the rate-versus-ionic strength curve is asymmetric, and that sliding can lead not only to acceleration, but also in some regimes to dramatic deceleration of the reaction.

Limits of analogy between self-avoidance and topology-driven swelling of polymer loops.

The work addresses the analogy between trivial knotting and excluded volume in looped polymer chains of moderate length, where the effects of knotting are small. A simple expression for the swelling seen in trivially knotted loops is described and shown to agree with simulation data. Contrast between this expression and the well-known expression for excluded volume polymers leads to a graphical mapping of excluded volume to trivial knots, which may be useful for understanding where the analogy between the two physical forms is valid. The work also includes description of a new method for the computational generation of polymer loops via conditional probability. Although computationally intensive, this method generates loops without statistical bias, and thus is preferable to other loop generation routines in the region of interest.

[A simple model of molecular construction]. / Prostaia model' molekuliarnoi konstruktsii.

The microscopic model illustrating the ideas of L.A. Blumenfeld about the role of slow conformational relaxation in the mechanism of action of enzymes is discussed.

Rational design of contact guiding, neurotrophic matrices for peripheral nerve regeneration.

Nerve guides filled with magnetically aligned hydrated gels of type I collagen have been shown to impart strong contact guidance cues to elongating neurites in vitro and to increase the number of regenerating axons in vivo relative to an isotropic collagen gel. We have formulated and analyzed a model to determine the conditions under which the target concentration of nerve growth factor (NGF) to support axonal growth can be sustained by entrapping either NGF-secreting cells or NGF-releasing polymer microspheres in the aligned gel. The equation describing NGF concentration with a distributed source term was solved after experimental determination of (1) the rate of NGF release from PLGA 85/15 microspheres, (2) the NGF diffusion coefficient in the gel and nerve guide membrane containing the gel, and (3) the maximum microsphere loading that does not compromise the magnetic alignment of collagen fibrils. We find that for a rat sciatic nerve, when using a 1 mm diameter nerve guide within a commercially available collagen membrane, the microsphere loading limit will prevent the construct's capacity to sustain the target NGF concentration of 1 ng/ml at two months when either wild type Schwann cells or PLGA 85/15 microspheres are used as the NGF source. This target concentration, however, will be maintained when transfected cells described in the literature to hypersecrete NGF are used, or when the microspheres are used if the permeability of the nerve guide membrane can be moderately decreased. For a human median nerve, when using a 5 mm diameter nerve guide within a commercially available membrane, the microspheres are capable of sustaining NGF concentrations above 1 ng/ml to at least 75 days without the need to decrease membrane permeability.

A few disconnected notes related to Levinthal paradox.

We estimate that the longest protein chain capable of exhaustive sampling of all its conformations within a millisecond is shorter than 15 residues. This reinforces the understanding of Levinthal paradox which emerged in the last decade, namely, that cooperative (all-or-none) character of folding and unfolding transition is indicative of the sequences selected such that reliable folding does not require exhaustive conformation sampling. The opinion is formulated that the discussions of Levinthal paradox should now fly to the new spheres.

Electrophoresis of a charge-inverted macroion complex: Molecular-dynamics study.

We have performed molecular-dynamics simulations to study the effect of an external electric field on a macroion in the solution of multivalent Z : 1 salt. To obtain plausible hydrodynamics of the medium, we explicitly make the simulation of many neutral particles along with ions. In a weak electric field, the macroion drifts together with the strongly adsorbed multivalent counterions along the electric field, in the direction proving inversion of the charge sign. The reversed mobility of the macroion is insensitive to the external field, and increases with salt ionic strength. The reversed mobility takes a maximal value at intermediate counterion valence. The motion of the macroion complex does not induce any flow of the neutral solvent away from the macroion, which reveals screening of hydrodynamic interactions at short distances in electrolyte solutions. A very large electric field, comparable to the macroion unscreened field, disrupts charge inversion by stripping the adsorbed counterions off the macroion.

Primary sequences of proteinlike copolymers: Levy-flight-type long-range correlations.

We consider the statistical properties of primary sequences of two-letter HP copolymers (H for hydrophobic and P for polar) designed to have water soluble globular conformations with H monomers shielded from water inside the shell of P monomers. We show, both by computer simulations and by exact analytical calculation, that for large globules and flexible polymers such sequences exhibit long-range correlations which can be described by Levy-flight statistics.

Free energy self-averaging in protein-sized random heteropolymers.

Current theories of heteropolymers are inherently macroscopic, but are applied to mesoscopic proteins. To compute the free energy over sequences, one assumes self-averaging--a property established only in the macroscopic limit. By enumerating the states and energies of compact 18, 27, and 36mers on a lattice with an ensemble of random sequences, we test the self-averaging approximation. We find that fluctuations in the free energy between sequences are weak, and that self-averaging is valid at the scale of real proteins. The results validate sequence design methods which exponentially speed up computational design and simplify experimental realizations.

Unexpected scenario of glass transition in polymer globules: an exactly enumerable model.

We introduce a lattice model of glass transition in polymer globules. This model exhibits ergodicity breaking in which the disjoint regions of phase space do not arise uniformly, but as small chambers whose number increases exponentially with polymer density. Chamber sizes obey power law distribution, making phase space similar to a fractal foam. This clearly demonstrates the importance of the phase space geometry and topology in describing any glass-forming system, such as semicompact polymers during protein folding.

Random walks in the space of conformations of toy proteins.

Monte Carlo dynamics of the lattice toy protein of 48 monomers is interpreted as a random walk in an abstract (discrete) space of conformations. To test the geometry of this space, we examine the return probability P(T), which is the probability to find the polymer in the native state after T Monte Carlo steps, provided that it starts from the native state at the initial moment. Comparing computational data with the theoretical expressions for P(T) for random walks in a variety of different spaces, we show that conformation spaces of polymer loops may have nontrivial dimensions and exhibit negative curvature characteristics of Lobachevskii (hyperbolic) geometry.

Gel catalysts that switch on and off.

We report development of a polymer gel with a catalytic activity that can be switched on and off when the solvent composition is changed. The gel consists of two species of monomers. The major component, N-isopropylacrylamide, makes the gel swell and shrink in response to a change in composition of ethanol/water mixtures. The minor component, vinylimidazole, which is capable of catalysis, is copolymerized into the gel network. The reaction rate for catalytic hydrolysis of p-nitrophenyl caprylate was small when the gel was swollen. In contrast, when the gel was shrunken, the reaction rate increased 5 times. The activity changes discontinuously as a function of solvent composition, thus the catalysis can be switched on and off by an infinitesimal change in solvent composition. The kinetics of catalysis by the gel in the shrunken state is well described by the Michaelis-Menten formula, indicating that the absorption of the substrate by the hydrophobic environment created by the N-isopropylacrylamide polymer in the shrunken gel is responsible for enhancement of catalytic activity. In the swollen state, the rate vs. active site concentration is linear, indicating that the substrate absorption is not a primary factor determining the kinetics. Catalytic activity of the gel is studied for substrates with various alkyl chain lengths; of those studied the switching effect is most pronounced for p-nitrophenyl caprylate.

Closed loops of nearly standard size: common basic element of protein structure.

By screening the crystal protein structure database for close Calpha-Calpha contacts, a size distribution of the closed loops is generated. The distribution reveals a maximum at 27+/-5 residues, the same for eukaryotic and prokaryotic proteins. This is apparently a consequence of polymer statistic properties of protein chain trajectory. That is, closure into the loops depends on the flexibility (persistence length) of the chain. The observed preferential loop size is consistent with the theoretical optimal loop closure size. The mapping of the detected unit-size loops on the sequences of major typical folds reveals an almost regular compact consecutive arrangement of the loops. Thus, a novel basic element of protein architecture is discovered; structurally diverse closed loops of the particular size.

Reversible molecular adsorption based on multiple-point interaction by shrinkable gels.

A general approach is presented for creating polymer gels that can recognize and capture a target molecule by multiple-point interaction and that can reversibly change their affinity to the target by more than one order of magnitude. The polymers consist of majority monomers that make the gel reversibly swell and shrink and minority monomers that constitute multiple-point adsorption centers for the target molecule. Multiple-point interaction is experimentally proven by power laws found between the affinity and the concentration of the adsorbing monomers within the gels.

Models of protein interactions: how to choose one.

BACKGROUND: There have been many attempts to approximate realistic protein interaction energies by coarse graining (i.e. considering interactions between amino acids rather than those between atoms). In particular, many 20-letter models have been derived (corresponding to the 20 naturally occurring amino acids). Because such models remain computationally infeasible, many two-letter models have been proposed as further simplifications. The choice of which model to use remains arbitrary, however. In this work, we formulate the framework within which the quality of approximate interaction potentials with respect to folding can be defined explicitly. RESULTS: Using a recently proposed criterion for comparing interaction matrices, we compare various 20 x 20 interaction matrices and obtain the two-letter model that most closely approximates each 20 x 20 matrix. We find that there are considerable differences among the 20 x 20 matrices. In particular, some matrices are much more similar to the hydrophobic model than others. Furthermore, we find that although the best two-letter approximation of a 20-letter model is a significantly better approximation than a random two-letter model, it is still a poor approximation of realistic protein interactions. CONCLUSIONS: The determination of the best two-letter approximations of various 20-letter models of protein interaction energies reveals the degree to which hydrophobic interactions dominate in each of the models and hence in proteins.