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In a large number of organisms covering all phyla, the mitochondrial respiratory chain harbors, in addition to the conventional elements, auxiliary proteins that confer adaptive metabolic plasticity. The alternative oxidase (AOX) represents one of the most studied auxiliary proteins, initially identified in plants. In contrast to the standard respiratory chain, the AOX mediates a thermogenic cyanide-resistant respiration; a phenomenon that has been of great interest for over 2 centuries in that energy is not conserved when electrons flow through it. Here we summarize centuries of studies starting from the early observations of thermogenicity in plants and the identification of cyanide resistant respiration, to the fascinating discovery of the AOX and its current applications in animals under normal and pathological conditions., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The spectral and photophysical properties of two four-ring alloxazine derivatives, naphtho[2,3-g]pteridine-2,4(1H,3H)-dione (1a) and 1,3-dimethylnaphtho[2,3-g]pteridine-2,4(1H,3H)-dione, (1b) were studied. The propensity of 1a for excited-state proton transfer reactions in the presence of acetic acid as a catalyst was also studied, showing no signature of the reaction occurring. In addition, quenching of 1a fluorescence by acetic acid was investigated. Singlet and triplet states and spectral data for 1a and 1b were calculated using density functional theory TD-DFT at B3LYP/6-31G(d) and UB3LYP levels. Finally, fluorescence lifetime imaging microscopy (FLIM) using 1a and 1b as fluorescence probes was applied to in vitro human red blood cells (RBCs) with and without tert-butyl hydroperoxide (TB) as an oxidising agent. To evaluate and compare the effects of 1a and 1b on the redox properties of RBCs, the fluorescence lifetime, amplitude and fractional intensities were calculated, and phasor plot analysis was performed. The results obtained show the appearance of a new proximal cluster in the phasor fingerprint of RBCs in the presence of 1b and a shorter fluorescence lifetime of RBCs in the presence of 1a., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mechanisms and kinetic of particle deposition at solid surfaces leading to the formation of self-assembled layers of controlled structure and density were reviewed. In the first part theoretical aspects were briefly discussed, comprising limiting analytical solutions for the linear transport under flow and diffusion. Methods of the deposition kinetics analysis for non-linear regimes affected by surface blocking were also considered. Characteristic monolayer formation times under diffusion and flow for the nanoparticle size range were calculated. In the second part illustrative experimental results obtained for micro- and nanoparticles were discussed. Deposition at planar substrates was analyzed with emphasis focused on the stability of layers and the release kinetics of silver particles. Applicability of the quartz microbalance measurements (QCM) for quantitative studies of nanoparticle deposition kinetic was also discussed. Except for noble metal and polymer particles, representative results for virus deposition at abiotic surfaces were analyzed. Final part of the review was devoted to nanoparticle corona formation at polymer carrier particles investigated by combination of the concentration depletion, AFM, SEM and the in situ electrokinetic method. It is argued that the results obtained for colloid particles can be used as reliable reference systems for interpretation of protein and other bioparticle deposition, confirming the thesis that simple is universal., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Adsorption kinetics of myoglobin molecules on mica and silica was studied using the atomic force microscopy (AFM), the colloid enhancement and the quartz microbalance (QCM) methods. Measurements were carried out for the NaCl concentration of 0.01 and 0.15 M as a function of pH comprising pH 7.4 stabilized by the PBS buffer. The electrophoretic mobility measurements enabled to derive the molecules zeta potential as a function of pH. The isoelectric point appearing at pH 5, is lower than that predicted from the theoretical calculations of the nominal dissociation charge. The AFM investigations confirmed that myoglobin molecules irreversibly adsorb at pH 3.5 yielding well-defined layers of single molecules. These layers were characterized using the colloid enhancement method involving polymer microparticles for pH range 3-9. The microparticle deposition kinetics was adequately interpreted in terms of a hybrid random sequential adsorption model. It is confirmed that the myoglobin layers exhibit a negligible zeta potential at pH equal to 5 in accordance with the electrophoretic mobility measurements. Analogous adsorption kinetic measurements were performed for the silica substrate using QCM and AFM. It is observed that myoglobin molecules irreversibly adsorb at pH 3.5 forming stable layers of single molecules. On the other hand, its adsorption kinetics at larger pHs was much slower exhibiting a poorly defined maximum coverage. This was attributed to aggregation of the myoglobin solutions due to their vanishing charge. The kinetic QCM runs were adequately interpreted in terms of a theoretical model combining the Smoluchowski aggregation theory with the convective diffusion mass transfer theory., (Copyright © 2020. Published by Elsevier B.V.)

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adsorption kinetics of human serum albumin (HSA) at a silica substrate was studied using the QCM-D and AFM methods. Measurements were performed at pH 3.5 for various bulk suspension concentrations and ionic strengths. The QCM experimental data were compared with the dry coverage of HSA derived from AFM and from the solution of the mass transfer equation. In this way, the dynamic hydration functions and water factors of HSA monolayers were quantitatively evaluated as a function of dry coverage for various ionic strengths. Using the hydration functions, the HSA adsorption runs derived from QCM-D measurements were converted to the dry coverage vs. the time relationships. In this way, the maximum coverage of irreversibly bound HSA molecules was determined. It was equal to 0.35 and 1.4 mg m -2 for NaCl concentration of 0.001 and 0.15 M, respectively. These results agree with previous experimental data derived by streaming potential measurements for mica and with theoretical modeling. Therefore, the side-on mechanism of HSA adsorption at silica sensor at pH 3.5 was confirmed. Also, a quantitative analysis of the desorption runs allowed one to calculate the binding energy of the reversibly bound HSA fraction. Beside significance to basic science, these results enable to develop a robust technique of preparing HSA monolayers at silica sensor of well-controlled coverage and molecule orientation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human serum albumin (HSA) adsorption on positively and negatively charged polystyrene microparticles was studied at various pHs and NaCl concentrations. Thorough electrophoretic mobility measurements were carried out that enabled to monitor in situ the progress of protein adsorption. The maximum coverage of irreversibly adsorbed HSA on microparticles was determined by different concentration depletion methods, one of them involving AFM imaging of residual molecules. An anomalous adsorption of HSA on the positive microparticles was observed at pH 3.5 where the maximum coverage attained 1.0mgm -2 for NaCl concentrations of 0.05M despite that the molecules were on average positively charged. For comparison, the maximum coverage of HSA on negatively charged microparticles was equal to 1.3mgm -2 at this pH and NaCl concentration. At pH 7.4 the maximum coverage on positive microparticles was equal to 2.1mgm -2 for 0.05M NaCl concentration. On the other hand, for negative microparticles, negligible adsorption of HSA was observed at pH 7.4 and 9.7. These experimental data were adequately interpreted in terms of the random sequential adsorption approach exploiting the bead model of the HSA molecule. Different orientations of adsorbed molecules, inert alia, the edge-on orientation prevailing for positively charged microparticles at pH 7.4, were confirmed. This was explained in terms of a heterogeneous charge distribution over the HSA molecule prevailing for a wide range of pHs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Colloid particle deposition was applied in order to characterize human serum albumin (HSA) monolayers on mica adsorbed under diffusion transport at pH 3.5. The surface concentration of HSA was determined by a direct AFM imaging of single molecules. The electrokinetic characteristics of the monolayers for various ionic strength were done by in situ streaming potential measurements. In this way the mean-field zeta potential of monolayers was determined. It was shown that the initially negative potential changed its sign for HSA surface concentrations above 2800μm(-2) that was interpreted as overcharging effect. The monolayers were also characterized by the colloid deposition method where negatively charged polystyrene particles, 810nm in diameter were used. The kinetics of particle deposition and their maximum coverage were determined as a function of the HSA monolayer surface concentration. An anomalous deposition of particles on substrates exhibiting a negative zeta potential was observed, which contradicts the mean-field theoretical predictions. This effect was quantitatively interpreted in terms of the random site sequential adsorption model. It was shown that efficient immobilization of particles only occurs at adsorption sites formed by three and more closely adsorbed HSA molecules. These results can be exploited as useful reference data for the analysis of deposition phenomena of bioparticles at protein monolayers that has practical significance for the regulation of the bioadhesive properties of surfaces., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Physicochemical characteristics of the plasmid KfrA protein in electrolyte solutions were done using a combination of dynamic light scattering (DLS), atomic force microscopy (AFM) and electrokinetic methods. The size of the protein was determined via the diffusion coefficient measurements using DLS. It was revealed from these measurements that the protein exists in an aggregated state composed of four molecules. The size of the protein was also determined via AFM imaging of single molecules adsorbed on mica from dilute solutions at pH=3.5. It was 10.6 nm in accordance with the value predicted for an aggregate composed of four monomers in a hexagonal configuration. The aggregation number was also confirmed by kinetics measurements carried out under diffusion controlled transport using AFM imaging of proteins. Further characteristics were acquired via KfrA adsorption on polystyrene latex particles (average size of 820 nm). The electrophoretic mobility of the latex and its zeta potential were determined as a function of the coverage of the protein. The maximum monolayer coverage for pH=3.5 was 1.2 mgm(-2). Additionally, from these measurements the effective charge of KfrA tetramer equal to 12 e (elementary charges) was predicted. The KfrA monolayer on latex was used to determine the isoelectric point of the protein, which was pH=4.5. As concluded, the procedures used in our work proved advantageous for a direct determination of aggregation processes and the effective charge if minor amounts of a protein are available., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The study demonstrates an application of the front-face fluorescence spectroscopy combined with multivariate regression methods to the analysis of fluorescent beer components. Partial least-squares regressions (PLS1, PLS2, and N-way PLS) were utilized to develop calibration models between synchronous fluorescence spectra and excitation-emission matrices of beers, on one hand, and analytical concentrations of riboflavin and aromatic amino acids, on the other hand. The best results were obtained in the analysis of excitation-emission matrices using the N-way PLS2 method. The respective correlation coefficients, and the values of the root mean-square error of cross-validation (RMSECV), expressed as percentages of the respective mean analytic concentrations, were: 0.963 and 14% for riboflavin, 0.974 and 4% for tryptophan, 0.980 and 4% for tyrosine, and 0.982 and 19% for phenylalanine.

Immuno-supported liquid membrane (immuno-SLM) extraction is a new technique that makes use of antibody (Ab)-antigen interactions as the "extraction force" to drive the mass transfer in a selective way. In immuno-SLM, anti-analyte (Ag) Abs are introduced into the acceptor phase of the SLM unit to trap the Ag that passes from the flowing donor through the SLM into the stagnant acceptor. The amount of immuno-extracted analyte (AbAg) is quantified by connecting the immuno-SLM unit on-line with a non-competitive heterogeneous fluorescence flow immunoassay (FFIA) that makes use of a fluorescein-labeled analyte tracer that titrates the residual excess of Ab present in the acceptor. A restricted access (RA) column is used for the separation of the two tracer fractions (Ag* and AbAg*) formed, and the eluted AbAg* fraction is measured downstream by a fluorescence detector. Factors influencing the optimum immuno-SLM extraction parameters, i.e., donor flow rate, extraction time and type of Ab, were investigated for immuno extraction of the model analyte atrazine. Immuno-SLM coupled to FFIA (immuno-SLM-FFIA) and FFIA alone were compared in terms of the assay sensitivities obtained and the sample matrix influence. The concentration at the mid-point of the calibration curve (IC(50)) was 16.0+/-1.4 and 36+/-16 microg/l, the limit of detection (LOD) was 2.0+/-1.1 and 20+/-10 microg/l, and the dynamic range was 2-100 and 20-500 microg/l atrazine for immuno-SLM-FFIA and FFIA, respectively. The matrix influence on the FFIA was significant in orange juice and surface water, whereas the influence was minor for immuno-SLM-FFIA with recoveries between 104% and 115% for 5 microg/l atrazine in tap water, orange juice and river water.

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT(1) receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000-1800 cm(-1) region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-beta strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT(1) blockade is associated with attenuation of collagen absorption in these lesions.