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.

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.

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.

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.

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.

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.

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.

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.

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.

[Toroidal globular state of circular DNA]. / O toroidal'nom globuliarnom sostoianii kol'tsevoi zamknutoi DNK.

The influence of torsional elasticity of the double helix on compactization and structure of circular DNA in a compact form is studied in the case when the compact (globular) particle has a torus shape. For closed circular DNA the topological invariant, linking number of two strains, yields strict connection between conformation of double helix, considered as a unifilar homopolymer, and elastic energy of torsional twisting. The contribution of torsional elasticity to free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superturn's density. Torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA in comparison with the case of linear DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.

[Several possible conformational states of a homogeneous elastic polymeric chain]. / O nekotorykh vozmozhnykh konformatisionnykh sostoianiiakh odnorodnoi uprugoi polimernoi tsepi.

A homogenous elastic macromolecule, the so-called persistent model is considered. Possible initiation of dense globular state in it with nematic orderliness of the chain elements is analysed. The entropy of such a system in an arbitrary state is calculated. It is shown that a relatively short rigid chain forms a thoroidal structure. With the chain elongation thor is increased, without changing its proportions. When the parameters are changed the thoroidal structure may turn into the conventional one-linkage one. Possible application of this model for solving the problem of trimeric package of nuclei acids is discussed in a few words.

[Theoretical model of homopolymer chain adsorption on a homogenous surface]. / Teoreticheskaia model' adsorbtsii gomopolimernoi tsepi na odnorodnoi poverkhnosti

On the basis of I. M. Lifshitz's theory the adsorption of the homopolymer on the homogenous surface is considered. It is shown that the attraction of monomers to the surface may lead not only to adsorption of the globule or coil but also to the emergence of a new peculiar phase--dislike globule without the fringe, all the monomers of which are stuck to the surface. A two-dimensional phase-diagram is plotted.

[Globule--substrate complex: several effects caused by the polymeric nature of the globule]. / Kompleks globuly s substratom: nekotorye éffekty obuslovlennye polimernoi prirodoi globuly

To anaylse possible role of globule polymeric nature in enzyme functioning, an equilibrium complex of homopolymeric liquidlike globule with a hard ball which models the substrate is considered. It is shown that the presence of linear memory in the globule may lead to an increase of the braking moment of forces affecting the substrate, to additional phase transitions concerned with a transition of the substrate of the globule centram to the perifery and vice versa etc. The shape of the globule surface adsorbed on the substrate is calculated.

[A community model of mutually learning neuronal nets]. / Model' soobshchestva iz vzaimno obuchaiushchikhsiia drug u druga neironnykh setei.

A model of a community is suggested whose members are formal neuron nets interacting by signals exchange. As a signal each net can emit an image formed by it when recognising the preceding signal. The emitted signal comes to the inputs of other nets and is used as their initial state for the recognition process. The collective dynamics of such model is discussed for the case of non-learning nets. Possible algorithm of mutual learning of the nets in them course of signals exchange is considered.

[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.

[A theory of heteropolymers with frozen random primary structure: properties of the globular state, coil-globule transitions and possible biophysical applications]. / K teorii geteropolimerov s zamorozhennoi besporiadochnoi pervichnoi strukturoi: svoistva globuliarnogo sostoianiia, perekhod tipa klubok-globula, vozmozhnye biofizicheskie prilozheniia.

A new approach to the problem of correspondence between primary structures of polypeptides and their tertiary structures is presented. This approach is based on the following statement of the problem: what will be the statistical properties of tertiary structures provided that the statistical properties of primary structures (e. g. the ratio of polar and unpolar residues) are given? It is discussed whether such statement is useful for the investigation of the prebiological evolution and protein folding. It is emphasized that the basic point here is to determine the configurational entropy of the chain, i. e., the number of foldings which lead to a given density distribution space. Hence, the auxiliary problem of the ideal heteropolymer collapse in the external field is considered. This problem is investigated numerically in the case when the field is localized in the small region in space. It is shown that such geometrical characteristics as the size of a globule are not sensitive to the details of primary structure when the chain is a random coil, and become sensitive to such details in the opposite globular case. These characteristics in the globular state can be described by the stable probability distribution function which does not depend upon chain length. Geometric characteristics of the chain exhibit especially strong dependence upon the primary structure in the region of coil-globule transition which is of the second order for the ensemble of chains.

[Collective properties of the mutual-learned neuronal net systems in the information field]. / Kollektivnye svoistva sistemy vzaimoobuchaiushchikhsia neironnykh setei v informatsionnom pole.

Model of neural networks system, in which networks interact by transmission and associative recognition of signals, is studied by computer simulation and qualitative approach. System behavior depends on the value of learning parameter epsilon, which determines the weight of writing in memory of each network every transmissible signal. Two different regimes are found: regime of auto-governed behavior, which depends only on initial networks characteristics, and regime of collective recognition of initial signal in form of a certain stable signals cycle. Analogy of this model and Aigen's hypercycle, the problem of creation of some new information in this model are discussed, too.

[Solid interactions in statistical physics of biopolymers]. / Ob''emnye vzaimodeistviia v statisticheskoi fizike biopolimerov.

A short survey is presented of the development of statistical physics of biological macromolecules and its modern state. The main attention is paid to the analysis of the manifestation of scale invariance and fractal properties of biopolymers--DNA and proteins. Phase transitions related to the phase structure of DNA are briefly analysed. A more detailed account is given of phase transitions in globular proteins, denaturation problem, two phases of the melted globule and the theory of heteropolymers included. Some unsolved problems of this field of science and its prospects are discussed.

[Elastic nature of a polymeric globule]. / O prirode uprugosti polimernoi globuly

Relative role of energetical and entropy contributions to the stresses which arise in the globule body after substrate absorption is considered. An integral solution is given for a simple model of the globule. An analysis of the result enables a conclusion that the entropy part is not small (it is large or of the same value) as compared with the energetical one, of only some peculiar globules have no evolutionary advantages.

[The theory of double-helical DNA packing: globular state of a homogeneous elastic macromolecule in a small volume]. / K teorii upakovki dvukhspiral'noi DNK: globuliarnoe sostoianie odnorodnoi uprugoi makromolekuly v polosti malogo ob''ema.

Globular state of persistent chain modelling DNA double helix in the cavity with unpermeable walls was studied. The effect of excluded volume was accounted for by Onsager method. For the most interesting case when the cavity size is much smaller than the persistent length, the globule is like a hoop wound on the cavity walls on the inside. Depending on the molecule thickness four limiting regions with quantitatively distinguished properties of the globule are possible. For all the regions the thickness of " hoops " along and across the walls is found, as well as both characteristics widths of orientational distribution of the chain elements.