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During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene- 1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., Spanish State Research Agency, SRA/ 10.13039/501100011033 (grants BIO2016-76640-R and PID2019.107515RB.C21), ERDF/ESF

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region., Spanish Ministry of Economy and Competitiveness (grant: BIO2016-76640-R), ANRS and the Vaccine Research Institute for the Investissements d'Avenir program to C.M. and by the European Fund for Research and Development from the European Union., Departamento de Química Física, Facultad de Ciencias, Universidad de Granada. Grupo FQM-171 "Biofísica y Biotecnología Molecular"

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR–NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches., Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain, Ministerio de Economía y Competitividad (grants BIO2016-76640-R and BIO2016-78020-R) y Fondo Europeo para el Desarrollo Regional (FEDER) de la Unión Europea, ANRS and the Vaccine Research Institute for the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-77

Electrochemical impedance spectroscopy (EIS) is a widely used tool for characterization of fuel cells and other electrochemical conversion systems. When applied to the on-line monitoring in the context of in-field applications, the disturbances, drifts and sensor noise may cause severe distortions in the evaluated spectra, especially in the low-frequency part. Failure to ignore the random effects can result in misinterpreted spectra and, consequently, in misleading diagnostic reasoning. This fact has not been often addressed in the research so far. In this paper, we propose an approach to the quantification of the spectral uncertainty, which relies on evaluating the uncertainty of the equivalent circuit model (ECM). We apply the computationally efficient variational Bayes (VB) method and compare the quality of the results with those obtained with the Markov chain Monte Carlo (MCMC) algorithm. Namely, MCMC algorithm returns accurate distributions of the estimated model parameters, while VB approach provides the approximate distributions. By using simulated and real data we show that approximate results provided by VB approach, although slightly over-optimistic, are still close to the more realistic MCMC estimates. A great advantage of the VB method for online monitoring is low computational load, which is several orders of magnitude lower compared to MCMC. The performance of VB algorithm is demonstrated on a case of ECM parameters estimation in a 6 cell solid oxide fuel cell (SOFC) stack. The complete numerical implementation for recreating the results can be found at https://repo.ijs.si/lznidaric/variational-bayes-supplementary-material., 28 pages, 18 figures. Submitted to: Applied Energy

A set of long-term tests (t ≥ 1000 h) have been carried out in fuel cell and electrolysis modes on typical Ni-YSZ//YSZ//LSCF-CGO cells. The degradation rates were found to be higher in electrolysis than in fuel cell operation. Post-test analyses have revealed that Sr diffusion and formation of SrZrO3 at YSZ/CGO interface occur mainly during electrolysis operation, whereas the process is very limited in fuel cell mode. As a consequence, LSCF destabilization is found to be not involved in the degradation of cell performances during fuel cell operation while it could explain the highest degradation rates recorded in electrolysis mode. An in-house multi-scale model has been used to interpret the role of the cell operating mode on the LSCF demixing mechanism. The simulations have shown that the electrolysis operation leads to a strong depletion of oxygen vacancies in LSCF material (while the fuel cell condition results in an increase in the concentration of oxygen vacancies). It has been proposed that the depletion in oxygen vacancies under electrolysis polarization could drive the Sr release from the structure, and in turn, could explain the experimental results. Based on this proposition, a possible mechanism for the LSCF destabilization and SrZrO3 formation is detailed.

This paper deals with the monitoring of the internal condition of a 6-cell solid oxide fuel cell stack during a 3600-hour test with short fuel starvation intervals. The relationship between change in condition and change of impedance spectra as well as the parameters of the equivalent circuit models are analysed. Tentative features indicating reduced fuel supply are identified. The analysis uses discrete random binary sequence, a stochastic broadband excitation signal that allows much shorter perturbation times. More than 600 electrochemical impedance spectroscopy curves and the associated equivalent circuit model parameters were analysed. High fuel utilisation interval is detected in two ways: (i) by violating the Z-HIT test in the low-frequency part (below ≈ 1 Hz) and (ii) significant change of the area-specific resistance parameter. The degradation after fuel cut-offs is clearly reflected in the change in area-specific resistance and the serial resistance. The numerical implementation of the proposed algorithms is available at: https://repo.ijs.si/pboskoski/py_eis.git .

In this work, an online natural aging estimation algorithm is developed, coupled with an Electrochemical Impedance Spectroscopy (EIS)-based diagnostic algorithm, to refine detection features extraction during Solid Oxide Fuel Cell (SOFC) stack operation and to predict its Remaining Useful Life (RUL). A combination of a lumped dynamic model along with features extracted from real-time EIS measurements is herein proposed for on-line applications. An Equivalent Circuit Model (ECM) is considered to identify parameters, such as ohmic and total resistance, that are coupled with an Area Specific Resistance (ASR) approach within the lumped model. The information derived from the EIS spectrum allows to estimate the voltage degradation over time along with its nominal behaviour. Indeed, the time trend of the identified parameters is proportional to the aging of the cell if no other abnormal condition occurs. This guarantees an on-line RUL estimation and a more robust diagnostic algorithm for fault detection and isolation. The approach has been applied to a 6-cells anode supported short stack tested for about 5000 h, and the related RUL estimation identified a critical issue on the middle cell, affecting its neighbours.

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14–2.2 μm) than the substrate S-adenosylmethionine (KD of 22–43 μm), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

Samples of antimony pentafluoride from different syntheses as well as commercial products were investigated by 19 F and 1 H NMR and compared. HF was identified as impurity and SbF 6 − specie is also present. Their amounts have been correlated through a method based on 19 F NMR.

The importance of heterogeneous catalysis in single-chamber solid oxide fuel cells (SC-SOFC) is universally recognized, but little studied. This work presents a thorough investigation of the catalytic activity of three Ni-YSZ half-cells in a well-described single-chamber reactor. One in-house electrolyte-supported and two commercially available anode-supported half-cells composed of anodes with thicknesses ranging from 50 μm to 1.52 mm are investigated. They are exposed to methane and oxygen gas mixtures within CH 4 :O 2 flow rate ratios ( R in ) of 0.8–2.0 and furnace temperatures of 600–800 °C. The conversion of methane always results in the formation of syngas species (H 2 and CO). However, their yields vary considerably based on the individual anode, the operating temperature, and R in . The SC-reactor design and the presence of hot-spots at the reactor entrance bring the methane and oxygen conversion rates well above the limit expected from experiments carried out with anode half-cells only. Major variations in the H 2 /CO ratio are observed. In lowering the temperature from 800 °C to 600 °C, it spreads from well below to well above the stoichiometric value of 2.0 expected for the partial oxidation reaction. To optimize the SC-SOFC any further, the findings stress the need to undertake even more catalytic studies of its electrode materials under actual structure and morphology as well as final reactor configuration.

To understand and tackle amyloid-related diseases, it is crucial to investigate the factors that modulate amyloid formation of proteins. Our previous studies proved that the N47A mutant of the α-spectrin SH3 (Spc-SH3) domain forms amyloid fibrils quickly under mildly acidic conditions. Here, we analyze how experimental conditions influence the kinetics of assembly and the final morphology of the fibrils. Early formation of curly fibrils occurs after a considerable conformational change of the protein and the concomitant formation of small oligomers. These processes are strongly accelerated by an increase in salt concentration and temperature, and to a lesser extent by a reduction in pH. The rate-limiting step in these events has a high activation enthalpy, which is significantly reduced by an increase in NaCl concentration. At low-to-moderate NaCl concentrations, the curly fibrils convert to straight and twisted amyloid fibrils after long incubation times, but only in the presence of soluble species in the mixture, which suggests that the curly fibrils and the twisted amyloid fibrils are diverging assembly pathways. The results suggest that the influence of environmental variables on protein solvation is crucial in determining the nucleation kinetics, the pathway of assembly, and the final fibril morphology.

We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (Tm) and unfolding enthalpy (ΔH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

The catalytic activity of single chamber solid oxide fuel cells (SC-SOFCs) with respect to hydrocarbon fuels induces a major overheating of the fuel cell, temperature variations along its length, and changes in the original fuel/air composition mainly over the anode component. This paper assesses the temperature gradients and the variations in performance along electrolyte-supported Ni-YSZ/YSZ/LSM cells fed with methane gas. The investigations are performed in a useful range of CH4/O2 ratios between 1.0 and 2.0, in which the furnace temperature and flow rate of methane–air mixtures are held constant at 700 °C and 450 sccm, respectively. Electrochemical impedance spectroscopy (EIS) is used to sense the temperature at the location where smaller size cathodes are positioned on the opposite side of a full-size anode. Due to temperature increases, cells always perform better when the small cathodes are located at the inlet as well as at a CH4/O2 ratio of 1.0. With an increase in ratio, the results show the presence of artefacts due to the use of an active LSM material for the combustion of methane, and open-type gas distribution plates for the single chamber reactor.

The catalytic activity of symmetrical LSM (La0.8Sr0.2MnO3) cells operated under single chamber solid oxide fuel cell (SC-SOFC) conditions was investigated for methane-to-oxygen ratios Rin between 1 and 2. The oxidation reactions over electrodes sintered at 1100 °C (LSM1100) and 1200 °C (LSM1200) were studied, and the effect of any combustion was followed through electrochemical impedance spectroscopy (EIS). The activity of the LSM1100 electrode increases with temperature. Above 700 °C, the conversion of the oxygen species may exceed 30%. As a consequence, oxygen depletion is occurring and a low frequency semicircle in the EIS spectra becomes predominant. An increase of the sintering temperature to 1200 °C leads to a decrease in the catalytic activity. A LSM1100 electrode deposited on a Julich half-cell proves to reach better performance at 600 °C than at 700 °C. On such complete cells, however, the catalytic combustion becomes much more complex than on a LSM cathode alone. We are thus proposing a comprehensive parameter, Rout, that is summarizing the processes inside the single chamber reactor.

During electrolysis of molten KF-2HF, strongly adherent fluorine bubbles are generated at the surface of carbon anode. The current was passing even if the horizontal anode was fully covered with gas film. The formation and growth of gas bubbles were studied by transient electrochemical techniques. It was observed that the fluorine bubbles do not have the spherical cap shape predicted by the classical theory. The curvature radius of the interface profile is not constant, the edge of the bubble being flat with a contact angle close to zero. The results are interpreted in the frame of a model which takes into account the predominant role of the interfacial properties.

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.

Let (M,g,σ) be a compact spin manifold of dimension n⩾2. Let λ1+(g) be the smallest positive eigenvalue of the Dirac operator in the metric g∈[g] conformal to g. We then define λmin+(M,[g],σ)=infg∈[g]λ1+(g)Vol(M,g)1/n. We show that 0

The new composition Li 0.6 + Ni 0.70 II O 2− was prepared by room temperature electrochemical insertion of lithium into X-ray-amorphous nickel-oxide-based film. The film undergoes a reversible electrochemical Li + insertion process which is accompanied by a net electrochromic effect.