A combination of electrochemistry and mass spectrometry to monitor the interaction of reactive species with supported lipid bilayers.

Reactive oxygen and nitrogen species (RONS), e.g. generated by cold physical plasma (CPP) or photodynamic therapy, interfere with redox signaling pathways of mammalian cells, inducing downstream consequences spanning from migratory impairment to apoptotic cell death. However, the more austere impact of RONS on cancer cells remains yet to be clarified. In the present study, a combination of electrochemistry and high-resolution mass spectrometry was developed to investigate the resilience of solid-supported lipid bilayers towards plasma-derived reactive species in dependence of their composition. A 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer was undisturbed by 200 µM H2O2 (control) but showed full permeability after CPP treatment and space-occupying oxidation products such as PoxnoPC, PAzePC, and POPC hydroperoxide were found. Electron paramagnetic resonance spectroscopy demonstrated the presence of hydroxyl radicals and superoxide anion/hydroperoxyl radicals during the treatment. In contrast, small amounts of the intramembrane antioxidant coenzyme Q10 protected the bilayer to 50% and LysoPC was the only POPC derivative found, confirming the membrane protective effect of Q10. Such, the lipid membrane composition including the presence of antioxidants determines the impact of pro-oxidant signals. Given the differences in membrane composition of cancer and healthy cells, this supports the application of cold physical plasma for cancer treatment. In addition, the developed model using the combination of electrochemistry and mass spectrometry could be a promising method to study the effect of reactive species or mixes thereof generated by chemical or physical sources.

Dynamical structure factor of strongly coupled ions in a dense quantum plasma.

The dynamical structure factor (DSF) of strongly coupled ions in dense plasmas with partially and strongly degenerate electrons is investigated. The main focus is on the impact of electronic correlations (nonideality) on the ionic DSF. The latter is computed by carrying out molecular dynamics (MD) simulations with a screened ion-ion interaction potential. The electronic screening is taken into account by invoking the Singwi-Tosi-Land-Sjölander approximation, and it is compared to the MD simulation data obtained considering the electronic screening in the random phase approximation and using the Yukawa potential. We find that electronic correlations lead to lower values of the ion-acoustic mode frequencies and to an extension of the applicability limit with respect to the wave-number of a hydrodynamic description. Moreover, we show that even in the limit of weak electronic coupling, electronic correlations have a nonnegligible impact on the ionic longitudinal sound speed. Additionally, the applicability of the Yukawa potential with an adjustable screening parameter is discussed, which will be of interest, e.g., for the interpretation of experimental results for the ionic DSF of dense plasmas.

Self-diffusion in two-dimensional quasimagnetized rotating dusty plasmas.

The self-diffusion phenomenon in a two-dimensional dusty plasma at extremely strong (effective) magnetic fields is studied experimentally and by means of molecular dynamics simulations. In the experiment the high magnetic field is introduced by rotating the particle cloud and observing the particle trajectories in a corotating frame, which allows reaching effective magnetic fields up to 3000 T. The experimental results confirm the predictions of the simulations: (i) superdiffusive behavior is found at intermediate timescales and (ii) the dependence of the self-diffusion coefficient on the magnetic field is well reproduced.

Structural characteristics of strongly coupled ions in a dense quantum plasma.

The structural properties of strongly coupled ions in dense plasmas with moderately to strongly degenerate electrons are investigated in the framework of the one-component plasma model of ions interacting through a screened pair interaction potential. Special focus is put on the description of the electronic screening in the Singwi-Tosi-Land-Sjölander (STLS) approximation. Different cross-checks and analyses using ion potentials obtained from ground-state quantum Monte Carlo data, the random phase approximation (RPA), and existing analytical models are presented for the computation of the structural properties, such as the pair distribution and the static structure factor, of strongly coupled ions. The results are highly sensitive to the features of the screened pair interaction potential. This effect is particularly visible in the static structure factor. The applicability range of the screened potential computed from STLS is identified in terms of density and temperature of the electrons. It is demonstrated that at r_{s}>1, where r_{s} is the ratio of the mean interelectronic distance to the Bohr radius, electronic correlations beyond RPA have a nonnegligible effect on the structural properties. Additionally, the applicability of the hypernetted chain approximation for the calculation of the structural properties using the screened pair interaction potential is analyzed employing the effective coupling parameter approach.

Standardization of allergen products: 2. Detailed characterization of GMP-produced recombinant Phl p 5.0109 as European Pharmacopoeia reference standard.

BACKGROUND: The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant allergens and validated assays for the quantification of major allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available allergen reference standard. METHODS: Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage. RESULTS: PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity. CONCLUSIONS: Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia allergen reference standard for grass pollen products.

Wave spectra of a strongly coupled magnetized one-component plasma: quasilocalized charge approximation versus harmonic lattice theory and molecular dynamics.

Two different approaches to the calculation of the wave spectra of magnetized strongly coupled liquid one-component plasmas are analzyed: the semianalytical quasilocalized charge approximation (QLCA) and the angle-averaged harmonic lattice (AAHL) theory. Both theories are benchmarked against the numerical evidence obtained from molecular dynamics simulations. It is found that not too far from the melting transition (Γ≳100), the AAHL theory is superior to the QLCA, while further away from the transition, the QLCA performs comparably to or better than the AAHL theory.

Magnetizing a complex plasma without a magnetic field.

We propose and demonstrate a concept that mimics the magnetization of the heavy dust particles in a complex plasma while leaving the properties of the light species practically unaffected. It makes use of the frictional coupling between a complex plasma and the neutral gas, which allows us to transfer angular momentum from a rotating gas column to a well-controlled rotation of the dust cloud. This induces a Coriolis force that acts exactly as the Lorentz force in a magnetic field. Experimental normal mode measurements for a small dust cluster with four particles show excellent agreement with theoretical predictions for a magnetized plasma.

Oscillation spectrum of a magnetized strongly coupled one-component plasma.

A first-principles study of the collective oscillation spectrum of a strongly correlated one-component plasma in a strong magnetic field is presented. The spectrum consists of six fundamental modes that are found to be in good agreement with results from the quasilocalized charge approximation. At high frequencies, additional modes are observed that include Bernstein-type oscillations and their transverse counterparts, which are of importance for the high-frequency optical and transport properties of these plasmas.

Theoretical description of spherically confined, strongly correlated Yukawa plasmas.

A theoretical description of the radial density profile for charged particles with Yukawa interaction in a harmonic trap is described. At strong Coulomb coupling shell structure is observed in both computer simulations and experiments. Correlations responsible for such shell structure are described here using a recently developed model based in density functional theory. A wide range of particle number, Coulomb coupling, and screening lengths is considered within the fluid phase. A hypernetted chain approximation shows the formation of shell structure, but fails to give quantitative agreement with Monte Carlo simulation results at strong coupling. Significantly better agreement is obtained within the hypernetted chain structure using a renormalized coupling constant, representing bridge function corrections.

Collective excitations of a spherically confined Yukawa plasma.

The complete spectrum of eigenmodes of a spherically confined Yukawa plasma is presented, based on first-principle molecular dynamics simulations. These results are compared with a recent fluid theory for the multipole modes of this system [H. Kählert and M. Bonitz, Phys. Rev. E 82, 036407 (2010)] and with the exact N-particle eigenmodes in the crystalline phase. Simulations confirm the existence of high-order modes found in cold fluid theory. We investigate the influence of screening, coupling, and friction on the mode spectra in detail. Good agreement between theory and simulation is found for weak to moderate screening and low-order modes. In addition, a number of new modes are observed which are missing in the fluid theory. The relations between the breathing mode in the fluid theory, simulation, and the crystal eigenmode are investigated in further detail.

Nonlinear magnetoplasmons in strongly coupled Yukawa plasmas.

The existence of plasma oscillations at multiples of the magnetoplasmon frequency in a strongly coupled two-dimensional magnetized Yukawa plasma is reported, based on extensive molecular dynamics simulations. These modes are the analogues of Bernstein modes which are renormalized by strong interparticle correlations. Their properties are theoretically explained by a dielectric function incorporating the combined effect of a magnetic field, strong correlations and finite temperature.

How spherical plasma crystals form.

The correlation buildup and the formation dynamics of the shell structure in a spherically confined one-component plasma are studied. Using Langevin dynamics simulations the relaxation processes and characteristic time scales and their dependence on the pair interaction and dissipation in the plasma are investigated. While in systems with Coulomb interaction (e.g., trapped ions) in a harmonic confinement shell formation starts at the plasma edge and proceeds inward, this trend is significantly weakened for dusty plasmas with Yukawa interaction. With a suitable change of the confinement conditions the crystallization scenario can be externally controlled.

Fluid modes of a spherically confined Yukawa plasma.

The normal modes of a three-dimensional Yukawa plasma in an isotropic harmonic confinement are investigated by solving the linearized cold fluid equations. The eigenmodes are found analytically and expressed in terms of hypergeometric functions. It is found that the mode frequencies solely depend on the dimensionless plasma parameter ξ=κR , where R is the plasma radius and κ is the inverse screening length. The eigenfrequencies increase monotonically with ξ and saturate in the limit ξ→∞ . Compared with the results in the Coulomb limit [D. H. E. Dubin, Phys. Rev. Lett. 66, 2076 (1991)10.1103/PhysRevLett.66.2076], we find an additional class of modes characterized by the number n which determines the number of radial nodes in the perturbed potential. These modes originate from the degenerate bulk modes of the Coulomb system. Analytical formulas for the eigenfrequencies are derived for limiting cases.

Theoretical description of Coulomb balls: fluid phase.

A theoretical description for the radial density profile of a finite number of identical charged particles confined in a harmonic trap is developed for application over a wide range of Coulomb coupling (or, equivalently, temperatures) and particle numbers. A simple mean-field approximation neglecting correlations yields a density profile which is monotonically decreasing with radius for all temperatures, in contrast to molecular dynamics simulations and experiments showing shell structure at lower temperatures. A more complete theoretical description including charge correlations is developed here by an extension of the hypernetted chain approximation, developed for bulk fluids, to the confined charges. The results reproduce all of the qualitative features observed in molecular dynamics simulations and experiments. These predictions are then tested quantitatively by comparison with benchmark Monte Carlo simulations. Quantitative accuracy of the theory is obtained by correcting the hypernetted chain approximation with a representation for the associated bridge functions.

Probability of metastable configurations in spherical three-dimensional Yukawa crystals.

Recently the occurrence probabilities of ground and metastable states of three-dimensional Yukawa clusters with 27 and 31 particles have been analyzed in dusty plasma experiments [D. Block, Phys. Plasmas 15, 040701 (2008)]. There it was found that, in many cases, the ground state appeared substantially less frequently than excited states. Here we analyze this question theoretically by means of molecular dynamics (MD) and Monte Carlo simulations and an analytical method based on the canonical partition function. We confirm that metastable states can occur with a significantly higher probability than the ground state. The results strongly depend on the screening parameter of the Yukawa interaction and the damping coefficient used in the MD simulations. The analytical method allows one to gain insight into the mechanisms being responsible for the occurrence probabilities of metastable states in strongly correlated finite systems.

Characterization of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy.

BACKGROUND: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. METHODS: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. RESULTS: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. CONCLUSIONS: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

Generation of a low immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a.

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.

FIA acid-base titrations with a new flow-through pH detector.

A pH-sensitive detector for flow-through potentiometry based on a graphite/quinhydrone composite electrode was applied for flow-injection analysis (FIA) titrations. Hydrochloric acid and acetic acid were titrated by injection of samples into a sodium hydroxide carrier solution. System conditions were optimised by variation of flow rate, injection volume and titrant concentration. The parameters sampling frequency, residence time and dispersion coefficient were determined. The evaluation of peak width (time between the two inflection points on each side of the peak), peak area and slope of the raising edge of the peak lead were studied with respect to their use for calibration. Hydrochloric acid and acetic acid could be titrated down to a concentration of 2 x 10(-4) mol L(-1) using 150-microL injection volumes, which is almost ten times lower than can be achieved using colour indicators and a spectrophotometric detection.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.