Anisotropic Particle Deposition Kinetics from Quartz Crystal Microbalance Measurements: Beyond the Sphere Paradigm.

Deposition kinetics of polymer particles characterized by a prolate spheroid shape on gold sensors modified by the adsorption of poly(allylamine) was investigated using a quartz crystal microbalance and atomic force microscopy. Reference measurements were also performed for polymer particles of a spherical shape and the same diameter as the spheroid shorter axis. Primarily, the frequency and dissipation shifts for various overtones were measured as a function of time. These kinetic data were transformed into the dependence of the complex impedance, scaled up by the inertia impedance, upon the particle size to the hydrodynamic boundary layer ratio. The results obtained for low particle coverage were interpolated, which enabled the derivation of Sauerbrey-like equations, yielding the real particle coverage using the experimental frequency or dissipation (bandwidth) shifts. Experiments carried out for a long deposition time confirmed that, for spheroids, the imaginary and real impedance components were equal to each other for all overtones and for a large range of particle coverage. This result was explained in terms of a hydrodynamic, lubrication-like contact of particles with the sensor, enabling their sliding motion. In contrast, the experimental data obtained for spheres, where the impedance ratio was a complicated function of overtones and particle coverage, showed that the contact was rather stiff, preventing their motion over the sensor. It was concluded that results obtained in this work can be exploited as useful reference systems for a quantitative interpretation of bioparticle, especially bacteria, deposition kinetics on macroion-modified surfaces.

The Relevance of Reperfusion Stroke Therapy for miR-9-3p and miR-9-5p Expression in Acute Stroke-A Preliminary Study.

Reperfusion stroke therapy is a modern treatment that involves thrombolysis and the mechanical removal of thrombus from the extracranial and/or cerebral arteries, thereby increasing penumbra reperfusion. After reperfusion therapy, 46% of patients are able to live independently 3 months after stroke onset. MicroRNAs (miRNAs) are essential regulators in the development of cerebral ischemia/reperfusion injury and the efficacy of the applied treatment. The first aim of this study was to examine the change in serum miRNA levels via next-generation sequencing (NGS) 10 days after the onset of acute stroke and reperfusion treatment. Next, the predictive values of the bioinformatics analysis of miRNA gene targets for the assessment of brain ischemic response to reperfusion treatment were explored. Human serum samples were collected from patients on days 1 and 10 after stroke onset and reperfusion treatment. The samples were subjected to NGS and then validated using qRT-PCR. Differentially expressed miRNAs (DEmiRNAs) were used for enrichment analysis. Hsa-miR-9-3p and hsa-miR-9-5p expression were downregulated on day 10 compared to reperfusion treatment on day 1 after stroke. The functional analysis of miRNA target genes revealed a strong association between the identified miRNA and stroke-related biological processes related to neuroregeneration signaling pathways. Hsa-miR-9-3p and hsa-miR-9-5p are potential candidates for the further exploration of reperfusion treatment efficacy in stroke patients.

The pathogenic m.8993 T > G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase.

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.

Quantifying Nanoparticle Layer Topography: Theoretical Modeling and Atomic Force Microscopy Investigations.

A comprehensive method consisting of theoretical modeling and experimental atomic force microscopy (AFM) measurements was developed for the quantitative analysis of nanoparticle layer topography. Analytical results were derived for particles of various shapes such as cylinders (rods), disks, ellipsoids, hemispheres (caps), etc. It was shown that for all particles, their root-mean-square (rms) parameter exhibited a maximum at the coverage about 0.5, whereas the skewness was a monotonically decreasing function of the coverage. This enabled a facile determination of the particle coverage in the layer, even if the shape and size were not known. The validity of the analytical results was confirmed by computer modeling and experimental data acquired by AFM measurements for polymer nanoparticle deposition on mica and silica. The topographical analysis developed in this work can be exploited for a quantitative characterization of self-assembled layers of nano- and bioparticles, e.g., carbon nanotubes, silica and noble metal particles, DNA fragments, proteins, vesicles, viruses, and bacteria at solid surfaces. The acquired results also enabled a proper calibration, in particular the determination of the measurement precision, of various electron and scanning probe microscopies, such as AFM.

5-Deazaalloxazine as photosensitizer of singlet oxygen and potential redox-sensitive agent.

Flavins are a unique class of compounds that combine the features of singlet oxygen generators and redox-dependent fluorophores. From a broad family of flavin derivatives, deazaalloxazines are significantly underdeveloped from the point of view of photophysical properties. Herein, we report photophysics of 5-deazaalloxazine (1a) in water, acetonitrile, and some other solvents. In particular, triplet excited states of 1a in water and in acetonitrile were investigated using ultraviolet-visible (UV-Vis) transient absorption spectroscopy. The measured triplet lifetimes for 1a were all on the microsecond time scale (≈ 60 µs) in deoxygenated solutions. The quantum yield of S1 → T1 intersystem crossing for 1a in water was 0.43 based on T1 energy transfer from 1a to indicaxanthin (5) acting as acceptor and on comparative actinometric measurements using benzophenone (6). 1a was an efficient photosensitizer for singlet oxygen in aerated solutions, with quantum yields of singlet oxygen in methanol of about 0.76, compared to acetonitrile ~ 0.74, dichloromethane ~ 0.64 and 1,2-dichloroethane ~ 0.54. Significantly lower singlet oxygen quantum yields were obtained in water and deuterated water (Ð¤Δ ~ 0.42 and 0.44, respectively). Human red blood cells (RBC) were used as a cell model to study the antioxidant capacity in vitro and cytotoxic activity of 1a. Fluorescence-lifetime imaging microscopy (FLIM) data were analyzed by fluorescence lifetime parameters and distribution for different parts of the emission spectrum. Comparison of multidimensional fluorescent properties of RBC under physiological-like and oxidative-stress conditions in the presence and absence of 1a suggests its dual activity as probe and singlet-oxygen generator and opens up a pathway for using FLIM to analyze complex intracellular behavior of flavin-like compounds. These new data on structure-property relationship contribute to the body of information required for a rational design of flavin-based tools for future biological and biochemical applications.

QCM-D Investigations of Anisotropic Particle Deposition Kinetics: Evidences of the Hydrodynamic Slip Mechanisms.

Deposition kinetics of positively charged polymer microparticles, characterized by prolate spheroid shape, at silica and gold sensors was investigated using the quartz microbalance (QCM) technique. Reference measurements were also performed for positively charged polymer particles of spherical shape and the same mass as the spheroids. Primarily, the frequency and bandwidth shifts for various overtones were measured as a function of time. It is shown that the ratio of these signals is close to unity for all overtones. These results were converted to the dependence of the frequency shift on the particle coverage, directly determined by atomic force microscopy and theoretically interpreted in terms of the hydrodynamic model. A quantitative agreement with experiments was attained considering particle slip relative to the ambient oscillating flow. In contrast, the theoretical results pertinent to the rigid contact model proved inadequate. The particle deposition kinetics derived from the QCM method was compared with theoretical modeling performed according to the random sequential adsorption approach. This allowed to assess the feasibility of the QCM technique to furnish proper deposition kinetics for anisotropic particles. It is argued that the hydrodynamic slip effect should be considered in the interpretation of QCM kinetic results acquired for bioparticles, especially viruses.

Deregulating mitochondrial metabolite and ion transport has beneficial effects in yeast and human cellular models for NARP syndrome.

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene has been associated with numerous cases of neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome, which are diseases known to result from abnormalities affecting mitochondrial energy transduction. We previously reported that an equivalent point mutation severely compromised proton transport through the ATP synthase membrane domain (FO) in Saccharomyces cerevisiae and reduced the content of cytochrome c oxidase (Complex IV or COX) by 80%. Herein, we report that overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) considerably increases Complex IV abundance and tricarboxylic acid-mediated substrate-level phosphorylation of ADP coupled to conversion of α-ketoglutarate into succinate in m.8993T>G yeast. Consistently in m.8993T>G yeast cells, the retrograde signaling pathway was found to be strongly induced in order to preserve α-ketoglutarate production; when Odc1p was overexpressed, this stress pathway returned to an almost basal activity. Similar beneficial effects were induced by a partial uncoupling of the mitochondrial membrane with the proton ionophore, cyanide m-chlorophenyl hydrazone. This chemical considerably improved the glutamine-based, respiration-dependent growth of human cytoplasmic hybrid cells that are homoplasmic for the m.8993T>G mutation. These findings shed light on the interdependence between ATP synthase and Complex IV biogenesis, which could lay the groundwork for the creation of nutritional or metabolic interventions for attenuating the effects of mtDNA mutations.

Mitochondria are physiologically maintained at close to 50 °C.

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, but a substantial proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain (RC) was fully functional, both in human embryonic kidney (HEK) 293 cells and primary skin fibroblasts. This differential was abolished in cells depleted of mitochondrial DNA or treated with respiratory inhibitors but preserved or enhanced by expressing thermogenic enzymes, such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at or slightly above 50 °C. In view of their potential consequences, these observations need to be further validated and explored by independent methods. Our study prompts a critical re-examination of the literature on mitochondria.

SLC13A3 variants cause acute reversible leukoencephalopathy and α-ketoglutarate accumulation.

OBJECTIVE: SLC13A3 encodes the plasma membrane Na+ /dicarboxylate cotransporter 3, which imports inside the cell 4 to 6 carbon dicarboxylates as well as N-acetylaspartate (NAA). SLC13A3 is mainly expressed in kidney, in astrocytes, and in the choroid plexus. We describe two unrelated patients presenting with acute, reversible (and recurrent in one) neurological deterioration during a febrile illness. Both patients exhibited a reversible leukoencephalopathy and a urinary excretion of α-ketoglutarate (αKG) that was markedly increased and persisted over time. In one patient, increased concentrations of cerebrospinal fluid NAA and dicarboxylates (including αKG) were observed. Extensive workup was unsuccessful, and a genetic cause was suspected. METHODS: Whole exome sequencing (WES) was performed. Our teams were connected through GeneMatcher. RESULTS: WES analysis revealed variants in SLC13A3. A homozygous missense mutation (p.Ala254Asp) was found in the first patient. The second patient was heterozygous for another missense mutation (p.Gly548Ser) and an intronic mutation affecting splicing as demonstrated by reverse transcriptase polymerase chain reaction performed in muscle tissue (c.1016 + 3A > G). Mutations and segregation were confirmed by Sanger sequencing. Functional studies performed on HEK293T cells transiently transfected with wild-type and mutant SLC13A3 indicated that the missense mutations caused a marked reduction in the capacity to transport αKG, succinate, and NAA. INTERPRETATION: SLC13A3 deficiency causes acute and reversible leukoencephalopathy with marked accumulation of αKG. Urine organic acids (especially αKG and NAA) and SLC13A3 mutations should be screened in patients presenting with unexplained reversible leukoencephalopathy, for which SLC13A3 deficiency is a novel differential diagnosis. ANN NEUROL 2019;85:385-395.

Myoglobin molecule charging in electrolyte solutions.

All atom molecular dynamic modeling was applied in order to determine water molecule and electrolyte ion concentration profiles around and inside the myoglobin molecule at various pH values. Significant penetration of counter ions into the molecule was confirmed. The electric potential distribution within and outside the molecule was quantitatively described using the non-linear Poisson-Boltzmann (PB) approach. Using this model, calculations were performed, yielding the surface and zeta potential for various physicochemical parameters, comprising pH, the electric permittivity, the ion penetration depth and the protein volume fraction (crowding effect). The theoretical results were used for the interpretation of experimental data acquired under different ionic strengths and temperatures by electrophoretic mobility measurements. It is confirmed that the experimental data are adequately reflected for acidic pH values by the non-linear PB model where the nominal molecule charge was calculated from the H++ model. The deviations occurring for larger pH values were accounted for by considering additional non-electrostatic interactions stemming from the van der Waals and ion-induced dipole forces. In this way, it is both experimentally and theoretically confirmed that the effective charge of the myoglobin molecule in electrolyte solutions is considerably smaller than the nominal, structure-based, predicted charge. As a result, under physiological conditions prevailing, e.g. in skeletal muscles, the effective charge of the myoglobin molecule should practically vanish. One can expect that the approach developed in this work can be applied for predicting charging mechanisms of other protein molecules characterized by an analogous charge vs. pH characteristic, e.g., the SARS-CoV-2 virus spike proteins, and for soft particles with pH responsive characteristics.

Distinctive Krebs cycle remodeling in iPSC-derived neural and mesenchymal stem cells.

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.

Human Serum Albumin Adsorption Kinetics on Silica: Influence of Protein Solution Stability.

Adsorption kinetics of human serum albumin (HSA) on silica substrates was studied using optical waveguide lightmode spectroscopy (OWLS) and quartz microbalance (QCM) techniques. Measurements were performed at pH 3.5, 5.6, and 7.4 for various bulk suspension concentrations and ionic strengths. The diffusion coefficient measurements showed that for pH 3.5 the HSA molecules are stable for NaCl concentrations from 10-3 to 0.15 M. This allowed us to precisely determine the mass transfer rate coefficients for the OWLS and QCM cells. The experimental data were adequately interpreted in terms of a hybrid random sequential adsorption model. The OWLS maximum coverage of HSA at pH 3.5, which is equal to 1.3 mg m-2, agrees with the QCM result and with previous results derived from streaming potential measurements. Thus, the results obtained at pH 3.5 served as reference data for the analysis of adsorption kinetics at higher pHs. In this way, it was confirmed that the adsorption kinetics of HSA molecules at pH 5.6 and 7.4 was considerably slower than at pH 3.5. This effect was attributed to aggregation of HSA solutions and interpreted in terms of a theoretical model combining the Smoluchowski aggregation theory with the convective diffusion mass transfer theory. New analytical equations were derived that can be used for the interpretation of other protein adsorption from unstable solutions.

Gold substrates of controlled roughness and electrokinetic properties formed by nanoparticle deposition.

The kinetics of positively charged gold nanoparticle self-assembly on oxidized silicon substrates (wafers) under diffusion-controlled transport was studied using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The latter technique allowed the roughness parameters of the monolayer (root mean square) to be determined as a function of the particle coverage. These results were adequately interpreted in terms of a theoretical model developed for surfaces covered by features of spherical shape considering the tip convolution effect. The stability and the electrokinetic characteristics (zeta potential) of the monolayers were also acquired using streaming potential measurements. It was shown that the inversion of the negative zeta potential of the bare substrate (overcharging) occurs at the particle coverage equal to 0.15, and for larger coverages positive zeta potential values were asymptotically attained. Additionally, the desorption kinetics of the particles was investigated by the streaming potential method, which confirmed the stability of the monolayers for a broad range of pHs. It was argued that these results enable to develop an efficient method for the preparation of gold sensors exhibiting a well-controlled surface roughness and electrostatic charge comprising both negative and positive values.

Mutations in CYC1, encoding cytochrome c1 subunit of respiratory chain complex III, cause insulin-responsive hyperglycemia.

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c1 subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c1, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c1 is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals' fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c1 stability and complex III activity.

Mitochondrial cytochrome c oxidase deficiency.

As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance of studying different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy.

Alternative oxidase expression in the mouse enables bypassing cytochrome c oxidase blockade and limits mitochondrial ROS overproduction.

Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.

Unsuspected task for an old team: succinate, fumarate and other Krebs cycle acids in metabolic remodeling.

Seventy years from the formalization of the Krebs cycle as the central metabolic turntable sustaining the cell respiratory process, key functions of several of its intermediates, especially succinate and fumarate, have been recently uncovered. The presumably immutable organization of the cycle has been challenged by a number of observations, and the variable subcellular location of a number of its constitutive protein components is now well recognized, although yet unexplained. Nonetheless, the most striking observations have been made in the recent period while investigating human diseases, especially a set of specific cancers, revealing the crucial role of Krebs cycle intermediates as factors affecting genes methylation and thus cell remodeling. We review here the recent advances and persisting incognita about the role of Krebs cycle acids in diverse aspects of cellular life and human pathology.

Cis-silencing of PIP5K1B evidenced in Friedreich's ataxia patient cells results in cytoskeleton anomalies.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease characterized by ataxia, variously associating heart disease, diabetes mellitus and/or glucose intolerance. It results from intronic expansion of GAA triplet repeats at the FXN locus. Homozygous expansions cause silencing of the FXN gene and subsequent decreased expression of the encoded mitochondrial frataxin. Detailed analyses in fibroblasts and neuronal tissues from FRDA patients have revealed profound cytoskeleton anomalies. So far, however, the molecular mechanism underlying these cytoskeleton defects remains unknown. We show here that gene silencing spreads in cis over the PIP5K1B gene in cells from FRDA patients (circulating lymphocytes and primary fibroblasts), correlating with expanded GAA repeat size. PIP5K1B encodes phosphatidylinositol 4-phosphate 5-kinase ß type I (pip5k1ß), an enzyme functionally linked to actin cytoskeleton dynamics that phosphorylates phosphatidylinositol 4-phosphate [PI(4)P] to generate phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Accordingly, loss of pip5k1ß function in FRDA cells was accompanied by decreased PI(4,5)P2 levels and was shown instrumental for destabilization of the actin network and delayed cell spreading. Knockdown of PIP5K1B in control fibroblasts using shRNA reproduced abnormal actin cytoskeleton remodeling, whereas over-expression of PIP5K1B, but not FXN, suppressed this phenotype in FRDA cells. In addition to provide new insights into the consequences of the FXN gene expansion, these findings raise the question whether PIP5K1B silencing may contribute to the variable manifestation of this complex disease.

Modular assembly of yeast mitochondrial ATP synthase.

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring. These studies show that assembly of the ATP synthase is not a single linear process, as previously thought, but rather involves two separate but coordinately regulated pathways that converge at the end stage.