H+-Selective Electrodes Based on Amine-Type Ionophores: Generalized Theory and A Priori Quantification of Lower and Upper Detection Limits.

A critical analysis of the known theories of functioning of H+-selective electrodes (H+-SEs) based on neutral amine-type carriers is given. A model of specific ion association is proposed, according to which, in membranes plasticized with 2-nitrophenyloctyl ether, the protonated ionophore and cation-exchanger form much stronger ion pairs with inorganic ions extracted from the sample solution than with each other, and simple equations that describe the lower and upper limit detection (pHUDL and pHLDL) are obtained. A feasible and reliable method for quantifying the pKa values of ionophores in the membrane phase from potentiometric data is substantiated. The efficiency of using single-ion partition coefficients and ion pair formation constants for a priori quantitative description of the H+-SE response in solutions of various compositions has been demonstrated for the first time. It is shown that the width of the dynamic response range of such electrodes depends on the nature of the tertiary amino group, and the reasons for the observed effect are discussed.

Matrix is everywhere: extracellular DNA is a link between biofilm and mineralization in Bacillus cereus planktonic lifestyle.

To date, the mechanisms of biomineralization induced by bacterial cells in the context of biofilm formation remain the subject of intensive studies. In this study, we analyzed the influence of the medium components on the induction of CaCO3 precipitation by the Bacillus cereus cells and composition of the extracellular matrix (ECM) formed in the submerged culture. While the accumulation of extracellular polysaccharides and amyloids appeared to be independent of the presence of calcium and urea during the growth, the accumulation of extracellular DNA (eDNA), as well as precipitation of calcium carbonate, required the presence of both ingredients in the medium. Removal of eDNA, which was sensitive to treatment by DNase, did not affect other matrix components but resulted in disruption of cell network formation and a sixfold decrease in the precipitate yield. An experiment with a cell-free system confirmed the acceleration of mineral formation after the addition of exogenous salmon sperm DNA. The observed pathway for the formation of CaCO3 minerals in B. cereus planktonic culture included a production of exopolysaccharides and negatively charged eDNA lattice promoting local Ca2+ supersaturation, which, together with an increase in the concentration of carbonate ions due to pH rise, resulted in the formation of an insoluble precipitate of calcium carbonate. Precipitation of amorphous CaCO3 on eDNA matrix was followed by crystal formation via the ACC-vaterite-calcite/aragonite pathway and further formation of larger mineral aggregates in complex with extracellular polymeric substances. Taken together, our data showed that DNA in extracellular matrix is an essential factor for triggering the biomineralization in B. cereus planktonic culture.

A feasible, fast and reliable method for estimating ion-site association constants in plasticized PVC ion-selective electrode membranes.

A feasible, fast and reliable method for estimating ion association constants in PVC plasticized membranes of ion-selective electrodes from potentiometric data has been theoretically and experimentally substantiated. The method is based on the established fact of complete dissociation of salts of quaternary ammonium cations R4N + An‒ (except for those containing methyl substituents at the nitrogen atom) in a membrane plasticized with o-nitrophenyl octyl ether (o-NPOE). Therefore, the boundary potential at the interface of the membrane with an aqueous solution of R4N+ depends only upon the concentrations of the corresponding solution and the ion exchanger in the membrane and is independent of the presence of a lipophilic ionic additive (LIA), which makes it possible to use such ions as reference ones in the internal filling solution. If the ions studied (i+) are capable of forming ion associates with the ion exchanger, then the introduction of LIA into the membrane will lead to a decrease in the concentration of free i+ ions and to a corresponding increase in the boundary potential, from which the ion association constant can be directly calculated. The results obtained agree with the known literature data and the results of quantum chemical calculations. The prospective of applying the proposed method to the study of other membrane compositions is discussed.

Determination of Single-Ion Partition Coefficients between Water and Plasticized PVC Membrane Using Equilibrium-Based Techniques.

An experimentally simple method for the direct determination of single-ion partition coefficients between water and a PVC membrane plasticized with o-NPOE is suggested. The method uses the traditional assumption of equal single-ion partition coefficients for some reference cation and anion, in this case tetraphenylphosphonium (TPP+) and tetraphenylborate (TPB-). The method is based on an integrated approach, including direct study of some salts' distribution between water and membrane phases, estimation of ion association constants, and measurements of unbiased selectivity coefficients for ions of interest, including the reference ones. The knowledge of distribution coefficients together with ion association constants allows for direct calculation of the multiple of the single-ion partition coefficients for the corresponding cation and anion, while the knowledge of unbiased selectivity coefficients together with ion association constants allows for immediate estimation of the single-ion partition coefficients for any ion under study, if the corresponding value for the reference ion is known. Both potentiometric and extraction studies are inherently equilibrium-based techniques, while traditionally accepted methods such as voltammetry and diffusion are kinetical. The inner coherent scale of single-ion partition coefficients between water and membrane phases was constructed.

Caught red handed: modeling and confirmation of the myeloperoxidase ceruloplasmin alpha-thrombin complex.

The work is devoted to the study of the structural characteristics of the myeloperoxidase-ceruloplasmin-thrombin complex using small-angle neutron scattering methods in combination with computer modeling, as well as surface plasmon resonance and solid-phase enzyme assay. We have previously shown that the functioning of active myeloperoxidase during inflammation, despite the presence in the blood of an excess of ceruloplasmin which inhibits its activity, is possible due to the partial proteolysis of ceruloplasmin by thrombin. In this study, the myeloperoxidase-ceruloplasmin-thrombin heterohexamer was obtained in vitro. The building of a heterohexamer full-atomic model in silico, considering the glycosylation of the constituent proteins, confirmed the absence of steric barriers for the formation of protein-protein contacts. It was shown that the partial proteolysis of ceruloplasmin does not affect its ability to bind to myeloperoxidase, and a structural model of the heterohexamer was obtained using the small-angle neutron scattering method.

Quantum-classical mechanics as an alternative to quantum mechanics in molecular and chemical physics.

In quantum mechanics, the theory of quantum transitions is grounded on the convergence of a series of time-dependent perturbation theory. In nuclear and atomic physics, this series converges because the dynamics of quantum transitions (quantum jumps) are absent by definition. In molecular and chemical physics, on the contrary, the dynamics of "quantum" transitions, being determined by the joint motion of a light electron (or electrons) and very heavy nuclei, are present by definition, and the series of time-dependent perturbation theory becomes singular. An exception is the dynamic problem for stationary states in the Born-Oppenheimer adiabatic approximation, when the electronic subsystem turns out to be "off" from the general dynamic process and therefore is not dynamically full-fledged: it only forms an electric potential in which the nuclei oscillate. Removing the aforementioned singularity can be accomplished in two ways. The first method was consisted of introducing an additional postulate in the form of the Franck-Condon principle into molecular quantum mechanics, in which the adiabatic approximation is used. The second method was proposed by the author and consisted of damping the singular dynamics of the joint motion of an electron and nuclei in the intermediate (transient) state of molecular "quantum" transitions by introducing chaos. This chaos arises only during molecular quantum transitions and is called dozy chaos. Formally, the damping is carried out by replacing an infinitely small imaginary addition in the spectral representation of the complete Green's function of the system with its finite quantity. The damping chaos (dozy chaos) leads to the continuity of the energy spectrum in the molecular transient state, which is a sign of classical mechanics. Meanwhile, the initial and final states of the molecule obey quantum mechanics in the adiabatic approximation. Molecular quantum mechanics, which takes into account the chaotic dynamics of the transient state of molecular "quantum" transitions, can be called quantum-classical (dozy-chaos) mechanics. The efficacy of the damping for the aforementioned singularity was previously shown by dozy-chaos mechanics of elementary electron transfers in condensed matter, which is the simplest case of dozy-chaos mechanics, and its applications to a whole number of problems, especially to the optical spectra in polymethine dyes and their aggregates. This paper provides a regular exposition of this dozy-chaos (quantum-classical) mechanics of the elementary electron transfers. The main results of its applications presented in the introduction are also described.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Overcoming of One More Pitfall in Boundary Element Calculations with Computer Simulations of Ion-Selective Electrode Response.

Computer simulations of ion-selective membrane electrodes using diffusion layer models based on finite-differences principle for calculating diffusion processes in both phases and taking into account the local ion exchange equilibrium at the interface are successfully used for clarifying and even predicting the influence of different diffusion factors on several time-dependent characteristics of electrodes. It is shown here that a well-established approach based on the assumption of the constant concentration of the interfering ion in the sample solution fails for solutions containing strongly interfering ions where the concentration of the interfering ion in the boundary layer of the solution can be far lower in comparison with its concentration in the bulk. The limitation is demonstrated by a drastic discrepancy between experimental and calculated curves for the dependence of potential on time. This limitation can be overcome by taking into account the change of the interfering ion concentration in the boundary layer in accordance with the electroneutrality condition. A good agreement between simulation results and experimental data is demonstrated.

Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein.

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils' detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.

A double-edged sword: supramolecular complexes of triazavirine display multicenter binding effects which influence aggregate formation.

Communicated by Ramaswamy H. Sarma.

Application of the interface equilibria-triggered dynamic diffusion model of the boundary potential for the numerical simulation of neutral carrier-based ion-selective electrodes response.

It is shown that a simple dynamic diffusion model of the boundary potential based on a separate, step-by-step, account of ion transfer across the membrane/aqueous solution interface and the diffusion processes within both phases which was proposed earlier for describing the response of ionophore-free membranes, can be successfully used for ionophore-based membranes as well. The model makes it possible to carry out both separate and joint account of the effects of co-extraction, transmembrane transport and ion exchange on the boundary potential and retains robustness in all the variants studied. The model adequately describes the ionophore-based electrode response over the entire range of concentrations and allows one to clearly demonstrate the dependence of lower detection limit on such parameters as the diffusion coefficients and the concentration of electroactive substances in the membrane phase, the thickness of the diffusion layer in the sample solution, the duration of the measurement, and the composition of the internal reference solution. The results of numerical simulation are in good agreement with the experimental data presented in the literature. As all the factors of influence considered above can easily be regulated in more or less wide limits, but at the same time, an estimation of their cumulative effect is not always possible on an intuitive level, the present model can be of practical interest for justifying the ways of optimizing the design of the ISE and the algorithm for performing measurements in solving specific analytical problems.

The amyloidogenicity of the influenza virus PB1-derived peptide sheds light on its antiviral activity.

The influenza virus polymerase complex is a promising target for new antiviral drug development. It is known that, within the influenza virus polymerase complex, the PB1 subunit region from the 1st to the 25th amino acid residues has to be is in an alpha-helical conformation for proper interaction with the PA subunit. We have previously shown that PB1(6-13) peptide at low concentrations is able to interact with the PB1 subunit N-terminal region in a peptide model which shows aggregate formation and antiviral activity in cell cultures. In this paper, it was shown that PB1(6-13) peptide is prone to form the amyloid-like fibrillar aggregates. The peptide homo-oligomerization kinetics were examined, and the affinity and characteristic interaction time of PB1(6-13) peptide monomers and the influenza virus polymerase complex PB1 subunit N-terminal region were evaluated by the SPR and TR-SAXS methods. Based on the data obtained, a hypothesis about the PB1(6-13) peptide mechanism of action was proposed: the peptide in its monomeric form is capable of altering the conformation of the PB1 subunit N-terminal region, causing a change from an alpha helix to a beta structure. This conformational change disrupts PB1 and PA subunit interaction and, by that mechanism, the peptide displays antiviral activity.

Triazavirine supramolecular complexes as modifiers of the peptide oligomeric structure.

In this study, we present molecular dynamics simulations of the antiviral drug triazavirine, that affects formation of amyloid-like fibrils of the model peptide (SI). According to our simulations, triazavirine is able to form linear supramolecular structures which can act as shields and prevent interactions between SI monomers. This model, as validated by simulations, provides an adequate explanation of triazavirine's mechanism of action as it pertains to SI peptide fibril formation.

An Interface Equilibria-Triggered Time-Dependent Diffusion Model of the Boundary Potential and Its Application for the Numerical Simulation of the Ion-Selective Electrode Response in Real Systems.

A simple dynamic model of the phase boundary potential of ion-selective electrodes is presented. The model is based on the calculations of the concentration profiles of the components in membrane and sample solution phases by means of the finite difference method. The fundamental idea behind the discussed model is that the concentration gradients in both membrane and sample solution phases determine only the diffusion of the components inside the corresponding phases but not the transfer across the interface. The transfer of the components across the interface at any time is determined by the corresponding local interphase equilibria. According to the presented model, each new calculation cycle begins with the correction of the components' concentrations in the near-boundary (first) layers of the membrane and solution, based on the constants of the interphase equilibria and the concentrations established at a given time as a result of diffusion. The corrected concentrations of the components in the boundary layers indicate the start of a new cycle every time with respect to the calculations of diffusion processes inside each phase from the first layer to the second one, and so on. In contrast to the well-known Morf's model, the above-mentioned layers do not comprise an imaginary part and are entirely localized in the corresponding phases, and this allows performing the calculations of the equilibrium concentrations by taking into account material balance for each component. The model remains operational for any realistic scenarios of the electrode functioning. The efficiency and predictive ability of the proposed model are confirmed by comparing the results of calculations with the experimental data on the dynamics of the potential change of a picrate-selective electrode in nitrate solutions when determining the selectivity coefficients using the methods recommended by IUPAC.

Nature of the optical band shapes in polymethine dyes and H-aggregates: dozy chaos and excitons. Comparison with dimers, H*- and J-aggregates.

Results on the theoretical explanation of the shape of optical bands in polymethine dyes, their dimers and aggregates are summarized. The theoretical dependence of the shape of optical bands for the dye monomers in the vinylogous series in line with a change in the solvent polarity is considered. A simple physical (analytical) model of the shape of optical absorption bands in H-aggregates of polymethine dyes is developed based on taking the dozy-chaos dynamics of the transient state and the Frenkel exciton effect in the theory of molecular quantum transitions into account. As an example, the details of the experimental shape of one of the known H-bands are well reproduced by this analytical model under the assumption that the main optical chromophore of H-aggregates is a tetramer resulting from the two most probable processes of inelastic binary collisions in sequence: first, monomers between themselves, and then, between the resulting dimers. The obtained results indicate that in contrast with the compact structure of J-aggregates (brickwork structure), the structure of H-aggregates is not the compact pack-of-cards structure, as stated in the literature, but a loose alternate structure. Based on this theoretical model, a simple general (analytical) method for treating the more complex shapes of optical bands in polymethine dyes in comparison with the H-band under consideration is proposed. This method mirrors the physical process of molecular aggregates forming in liquid solutions: aggregates are generated in the most probable processes of inelastic multiple binary collisions between polymethine species generally differing in complexity. The results obtained are given against a background of the theoretical results on the shape of optical bands in polymethine dyes and their aggregates (dimers, H*- and J-aggregates) previously obtained by V.V.E.

Improved separate solution method for determination of low selectivity coefficients.

Simple, fast, and theoretically substantiated experimental method for determination of improved selectivity coefficients is proposed. The method is based on the well-known fact that low selectivity coefficients determined by the separate solution method (SSM) are time-dependent and, upon our finding, this dependence is a well-defined linear function of time raised to the certain negative power. In particular, the selectivity coefficients obtained for equally charged primary and foreign ions by SSM linearly depend on time to the minus one-fourth. It was found that extrapolation of experimental data using this function to the intersection with Y axes gives reliable values of rather low selectivity coefficients (down to n × 10(-7)), which strongly differ from those measured using SSM and correspond well with the values obtained using the modified separate solution method (MSSM) proposed by Bakker. At the same time, the new method is free of one very essential limitation inherent to MSSM, namely, it is applicable after the conditioning of electrodes in the primary ion solution and can be repeated many times.

A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis.

The fibrillogenesis of a peptide corresponding to residues 35-51 of human α-lactalbumin (¹GYDTQAIVENNESTEYG¹7) can be dramatically enhanced by the addition of a tetrapeptide TDYG homologous to its C-terminus (TEYG). Generation of spontaneous hydrolytic products similar to this peptide was demonstrated by mass-spectrometry analysis of GYDTQAIVENNESTEYG peptide solution components during fibrillogenesis. Possible mechanisms and roles of short peptides in protein metabolism are discussed.

Amyloidogenic peptide homologous to fragment 129-148 of human myocilin.

Myocilin is a protein with a molecular weight near 50 kDa. It is expressed in almost all organs and tissues. We showed that the peptide DQLETQTRELETAYSNLLRD corresponding to N-terminal Leucine zipper motif (LZM) of the protein is able to form amyloid-like fibrils. The possible role of this motif in myocilin aggregation is discussed.

Structural Features of the Peptide Homologous to 6-25 Fragment of Influenza A PB1 Protein.

A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.

Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1.

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.