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In this study, a detailed structural characterization of epitaxial La0.6Sr0.4CoO3-δ (LSC) films grown in (100), (110), and (111) orientations was conducted. LSC is a model air electrode material in solid oxide fuel and electrolysis cells and understanding the correlation of bulk structure and catalytic activity is essential for the design of future electrode materials. Thin films were grown on single crystals of the perovskite material La0.95Sr0.05Ga0.95Mg0.05O3-δ cut in three different directions. This enabled an examination of structural details at the atomic scale for a realistic material combination in solid oxide cells. The investigation involved the application of atomic force microscopy, X-ray diffraction, and high-resolution transmission electron microscopy to explore the distinct properties of these thin films. Interestingly, ordering phenomena in both cationic as well as anionic sublattices were found, despite the fact that the thin films were never at higher temperatures than 600 °C. Cationic ordering was found in spherical precipitates, whereas the ordering of oxygen vacancies led to the partial transition to brownmillerite in all three orientations. Our results indicate a very high oxygen vacancy concentration in all three thin films. Lattice strains in-plane and out-of-plane was measured, and its implications for the structural modifications are discussed.

Improving materials for energy conversion and storage devices is deeply connected with an optimization of their surfaces and surface modification is a promising strategy on the way to enhance modern energy technologies. This study shows that surface modification with ultra-thin oxide layers allows for a systematic tailoring of the surface dipole and the work function of mixed ionic and electronic conducting oxides, and it introduces the ionic potential of surface cations as a readily accessible descriptor for these effects. The combination of X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) illustrates that basic oxides with a lower ionic potential than the host material induce a positive surface charge and reduce the work function of the host material and vice versa. As a proof of concept that this strategy is widely applicable to tailor surface properties, we examined the effect of ultra-thin decoration layers on the oxygen exchange kinetics of pristine mixed conducting oxide thin films in very clean conditions by means of in-situ impedance spectroscopy during pulsed laser deposition (i-PLD). The study shows that basic decorations with a reduced surface work function lead to a substantial acceleration of the oxygen exchange on the surfaces of diverse materials., (© 2024. The Author(s).)

Spinels of the general formula Li 2-δ M 2 O 4 are an essential class of cathode materials for Li-ion batteries, and their optimization in terms of electrode potential, accessible capacity, and charge/discharge kinetics relies on an accurate understanding of the underlying solid-state mass and charge transport processes. In this work, we report a comprehensive impedance study of sputter-deposited epitaxial Li 2-δ Mn 2 O 4 thin films as a function of state-of-charge for almost the entire tetrahedral-site regime (1 ≤ δ ≤ 1.9) and provide a complete set of electrochemical properties, consisting of the charge-transfer resistance, ionic conductivity, volume-specific chemical capacitance, and chemical diffusivity. The obtained properties vary by up to three orders of magnitude and provide essential insights into the point defect concentration dependences of the overall electrode potential. We introduce a defect chemical model based on simple concentration dependences of the Li chemical potential, considering the tetrahedral and octahedral lattice site restrictions defined by the spinel crystal structure. The proposed model is in excellent qualitative and quantitative agreement with the experimental data, excluding the two-phase regime around 4.15 V. It can easily be adapted for other transition metal stoichiometries and doping states and is thus applicable to the defect chemical analysis of all spinel-type cathode materials., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The oxygen exchange kinetics and the surface chemistry of epitaxially grown, dense La 0.6 Sr 0.4 CoO 3-δ (LSC) thin films in three different orientations, (001), (110), and (111), were investigated by means of in situ impedance spectroscopy during pulsed laser deposition (i-PLD) and near-ambient-pressure X-ray photoelectron spectroscopy (NAP-XPS). i-PLD measurements showed that pristine LSC surfaces exhibit very fast surface exchange kinetics but revealed no significant differences between the specific orientations. However, as soon as the surfaces were in contact with acidic, gaseous impurities, such as S-containing compounds in nominally pure measurement atmospheres, NAP-XPS measurements revealed that the (001) orientation is substantially more susceptible to the formation of sulfate adsorbates and a concomitant performance decrease. This result is further substantiated by a stronger increase of the work function on (001)-oriented LSC surfaces upon sulfate adsorbate formation and by a faster performance degradation of these surfaces in ex situ measurement setups. This phenomenon has potentially gone unnoticed in the discussion of the interplay between the crystal orientation and the oxygen exchange kinetics and might have far-reaching implications for real solid oxide cell electrodes, where porous materials exhibit a wide variety of differently oriented and reconstructed surfaces., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The oxygen exchange kinetics of epitaxial Pr 0.1 Ce 0.9 O 2-δ electrodes was modified by decoration with submonolayer amounts of different basic (SrO, CaO) and acidic (SnO 2 , TiO 2 ) binary oxides. The oxygen exchange reaction (OER) rate and the total conductivity were measured by in situ PLD impedance spectroscopy ( i -PLD), which allows to directly track changes of electrochemical properties after each deposited pulse of surface decoration. The surface chemistry of the electrodes was investigated by near-ambient pressure XPS measurements (NAP-XPS) at elevated temperatures and by low-energy ion scattering (LEIS). While a significant alteration of the OER rate was observed after decoration with binary oxides, the pO 2 dependence of the surface exchange resistance and its activation energy were not affected, suggesting that surface decorations do not alter the fundamental OER mechanism. Furthermore, the total conductivity of the thin films does not change upon decoration, indicating that defect concentration changes are limited to the surface layer. This is confirmed by NAP-XPS measurements which find only minor changes of the Pr-oxidation state upon decoration. NAP-XPS was further employed to investigate changes of the surface potential step on decorated surfaces. From a mechanistic point of view, our results indicate a correlation between the surface potential and the altered oxygen exchange activity. Oxidic decorations induce a surface charge which depends on their acidity (acidic oxides lead to a negative surface charge), affecting surface defect concentrations, any existing surface potential step, potentially adsorption dynamics, and consequently also the OER kinetics.

The epiphytic orchid Caularthron bilamellatum sacrifices its water storage tissue for nutrients from the waste of ants lodging inside its hollow pseudobulb. Here, we investigate whether fungi are involved in the rapid translocation of nutrients. Uptake was analysed with a 15 N labelling experiment, subsequent isotope ratio mass spectrometry (IRMS) and secondary ion mass spectrometry (ToF-SIMS and NanoSIMS). We encountered two hyphae types: a thick melanized type assigned to 'black fungi' (Chaetothyriales, Cladosporiales, and Mycosphaerellales) in ant waste, and a thin endophytic type belonging to Hypocreales. In few cell layers, both hyphae types co-occurred. 15 N accumulation in both hyphae types was conspicuous, while for translocation to the vessels only Hypocreales were involved. There is evidence that the occurrence of the two hyphae types results in a synergism in terms of nutrient uptake. Our study provides the first evidence that a pseudobulb (=stem)-born endophytic network of Hypocreales is involved in the rapid translocation of nitrogen from insect-derived waste to the vegetative and reproductive tissue of the host orchid. For C. bilamellatum that has no contact with the soil, ant waste in the hollow pseudobulbs serves as equivalent to soil in terms of nutrient sources., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

The effects of sulphur adsorbates and other typical solid oxide fuel cell (SOFC) poisons on the electronic and ionic properties of an SrO-terminated (La,Sr)CoO 3 (LSC) surface and on its oxygen exchange kinetics have been investigated experimentally with near ambient pressure X-ray photoelectron spectroscopy (NAP-XPS), low energy ion scattering (LEIS) and impedance spectroscopy as well as computationally with density functional theory (DFT). The experiment shows that trace amounts of sulphur in measurement atmospheres form SO 2- 4 adsorbates and strongly deactivate a pristine LSC surface. They induce a work function increase, indicating a changing surface potential and a surface dipole. DFT calculations reveal that the main participants in these charge transfer processes are not sub-surface transition metals, but surface oxygen atoms. The study further shows that sulphate adsorbates strongly affect oxygen vacancy formation energies in the LSC (sub-)surface, thus affecting defect concentrations and oxygen transport properties. To generalize these results, the investigation was extended to other acidic oxides which are technologically relevant as SOFC cathode poisons, such as CO 2 and CrO 3 . The results unveil a clear correlation of work function changes and redistributed charge with the Smith acidity of the adsorbed oxide and clarify fundamental mechanistic details of atomic surface modifications. The impact of acidic adsorbates on various aspects of the oxygen exchange reaction rate is discussed in detail., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Electrochemical impedance spectroscopy was used to investigate the chemical capacitance of La 0.6 Sr 0.4 CoO 3-δ (LSC) thin-film electrodes under anodic polarization (i.e., in the electrolysis mode). For this purpose, electrodes with different microstructures were prepared via pulsed-laser deposition. Analysis of dense electrodes and electrodes with open porosity revealed decreasing chemical capacitances with increasing anodic overpotentials, as expected from defect chemical considerations. However, extremely high chemical capacitance peaks with values in the range of 10 4  F/cm 3 at overpotentials of >140 mV were obtained after annealing for several hours in synthetic air and/or after applying high anodic bias voltages of >750 mV. From the results of several surface analysis techniques and transmission electron microscopy, it is concluded that closed pores develop upon both of these treatments: (i) During annealing, initially open pores get closed by SrSO 4 , which forms due to strontium segregation in measurement gases with minute traces of sulfur. (ii) The bias treatment causes mechanical failure and morphological changes including closed pores in the bulk of dense films. Under anodic polarization, high-pressure oxygen accumulates in those closed pores, and this causes the capacitance peak. Model calculations based on a real-gas equation allow us to properly predict the experimentally obtained capacitance increase. We demonstrate that analysis of the chemical capacitance of oxygen electrodes in solid oxide electrolysis cells can thus be used as a nondestructive observation tool to detect and quantify closed porosity with a lower detection limit between 10 -4 and 10 -3 .

Minimizing the overpotential at the air electrode of solid oxide fuel cells (SOFC) is one of the key challenges regarding a broad applicability of this technology. Next to novel materials and geometry optimization, surface modification is a promising and flexible method to alter the oxygen exchange kinetics at SOFC cathode surfaces. Despite extensive research, the mechanism behind the effect of surface decorations is still under debate. Moreover, for Sr decoration, previous studies yielded conflicting results, reporting either a beneficial or a detrimental impact on the oxygen exchange kinetics. In this contribution, in situ impedance spectroscopy during pulsed laser deposition was used to investigate the effect of Sr containing decorations under different deposition conditions. Depending on deposition temperature and interactions with the gas phase, opposing effects of Sr decoration were found. In combination with near-ambient pressure X-ray photoelectron spectroscopy and non-ambient X-ray diffractometry, it was possible to trace this phenomenon back to different chemical environments of the surface Sr. At high temperatures, Sr is deposited as SrO, which can have a beneficial effect on the oxygen exchange kinetics. At low temperatures, SrCO 3 adsorbates are formed from trace amounts of CO 2 in the measurement atmosphere, causing a decrease of the oxygen exchange rate. These results are in excellent agreement with the concept of surface acidity as a descriptor for the effect of surface decorations, providing further insight into the oxygen exchange kinetics on SOFC cathode surfaces and its degradation. In addition, this study shows that Sr segregation itself initially does not lead to performance degradation but that segregated SrO readily reacts with acidic compounds, reducing the catalytic capability of mixed conducting oxides., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

La 0.6 Sr 0.4 FeO 3- δ (LSF) electrodes were grown on different electrolyte substrates by pulsed laser deposition (PLD) and their oxygen exchange reaction (OER) resistance was tracked in real-time by in situ PLD impedance spectroscopy (i-PLD) inside the PLD chamber. This enables measurements on pristine surfaces free from any contaminations and the direct observation of thickness dependent properties. As substrates, yttria-stabilized zirconia single crystals (YSZ) were used for polycrystalline LSF growth and La 0.95 Sr 0.05 Ga 0.95 Mg 0.05 O 3- δ (LSGM) single crystals or YSZ single crystals with a 5 nm buffer-layer of Gd 0.2 Ce 0.8 O 2- δ for epitaxial LSF film growth. While polycrystalline LSF electrodes show a constant OER resistance in a broad thickness range, epitaxially grown LSF electrodes exhibit a continuous and strong increase of the OER resistance with film thickness until ≈60 nm. In addition, the activation energy of the OER resistance increases by 0.23 eV compared to polycrystalline LSF. High resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) measurements reveal an increasing contraction of the out-of-plane lattice parameter in the epitaxial LSF electrodes over electrode thickness. Defect thermodynamic simulations suggest that the decrease of the LSF unit cell volume is accompanied by a lowering of the oxygen vacancy concentration, explaining both the resistive increase and the increased activation energy.

The chemical capacitance of La 0.6 Sr 0.4 CoO 3-δ (LSC) thin film microelectrodes with different microstructures was investigated upon varying anodic DC voltages. Dense and porous electrodes (open porosity) were prepared by using different parameters during pulsed laser deposition (PLD). Furthermore, electrodes with closed porosity were fabricated by depositing a dense capping layer on a porous film. Electrochemical impedance spectroscopy (EIS) was performed in synthetic air at 460 and 608 °C with anodic DC voltages up to 440 mV. Chemical capacitance values of the electrodes were derived from the obtained spectra. While the chemical capacitance of dense and porous electrodes decreased as expected with increasing anodic overpotential, electrodes with closed pores exhibited very unusual peaks with extremely high values of >8000 F/cm 3 at overpotentials of >100 mV. We demonstrate that this huge capacitance increase agrees very well with calculated chemical capacitances deduced from a real gas equation. Hence, we conclude that the formation of highly pressurized oxygen (up to gas pressures of ∼10 4  bar) in closed pores is responsible for this strong capacitive effect at anodic overpotentials. Such measurements can thus detect and quantify the buildup of high internal gas pressures in closed pores at the anode side of solid oxide electrolysis cells., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

In this study, five different mixed conducting cathode materials were grown as dense thin films by pulsed laser deposition (PLD) and characterized via in situ impedance spectroscopy directly after growth inside the PLD chamber (i-PLD). This technique enables quantification of the oxygen reduction kinetics on pristine and contaminant-free mixed conducting surfaces. The measurements reveal excellent catalytic performance of all pristine materials with polarization resistances being up to two orders of magnitude lower than those previously reported in the literature. For instance, on dense La 0.6 Sr 0.4 CoO 3- δ thin films, an area specific surface resistance of ∼0.2 Ω cm 2 at 600 °C in synthetic air was found, while values usually >1 Ω cm 2 are measured in conventional ex situ measurement setups. While surfaces after i-PLD measurements were very clean, ambient pressure X-ray photoelectron spectroscopy (AP-XPS) measurements found that all samples measured in other setups were contaminated with sulfate adsorbates. In situ impedance spectroscopy during AP-XPS revealed that already trace amounts of sulfur present in high purity gases accumulate quickly on pristine surfaces and lead to strongly increased surface polarization resistances, even before the formation of a SrSO 4 secondary phase. Accordingly, the inherent excellent catalytic properties of this important class of materials were often inaccessible so far. As a proof of concept, the fast kinetics observed on sulfate-free surfaces were also realized in ex situ measurements with a gas purification setup and further reduces the sulfur concentration in the high purity gas., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

This study focused on the total mercury content in fish from seven drinking-water reservoirs located in the Morava River Basin: Bojkovice, Boskovice, Hubenov, Karolinka, Landstejn, Ludkovice and Nova Rise in the Czech Republic. A total of 308 fish were collected for the analysis. The content of total mercury was measured in the muscle tissue of bream, roach and perch using atomic absorption spectrometry and varied from 0.057±0.009 to 0.440 mg kg -1 in bream, from 0.030±0.005 to 0.393±0.138 mg kg -1 in roach and from 0.092±0.007 to 0.638±0.042 mg kg -1 in perch. The highest total mercury content was found in perch from Landstejn and the lowest was measured in roach from Ludkovice. A positive statistically significant relationship was found between fish weight and total mercury content in fish muscle for almost all species and all sampling sites, except for roach from Ludkovice. A total of 19 samples exceeded the maximum mercury level set by legislation on food contaminants-0.5 mg kg -1 in freshwater fish., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Nominally undoped SrTiO 3 single crystals were illuminated by UV light at 350 °C in oxidizing as well as reducing atmospheres. In N 2 /O 2 atmospheres, UV irradiation enhances the conductivity of SrTiO 3 by several orders of magnitude. In dry H 2 atmosphere UV exposure leads to the opposite conductivity effect, i.e. , above band gap energy illumination surprisingly lowers the conductivity. This is discussed in the framework of a defect chemical model. We show that a shift in defect concentrations due to UV-driven oxygen incorporation from the gas phase into the oxide is the main cause of the measured conductivity changes. A model is introduced to illustrate the thermodynamic and kinetic drivers of the processes under UV irradiation. Noteably, in reducing H 2 /H 2 O atmospheres, the incorporation of oxygen into the investigated oxide under UV light takes place via water splitting. Owing to the predominant electron conduction of SrTiO 3 in equilibrium with H 2 , oxygen incorporation upon UV and thus an increase of the oxygen chemical potential leads to a decrease of the majority electronic charge carrier, here electrons, which lowers the conductivity under UV irradiation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Accelerating the oxygen reduction kinetics of solid oxide fuel cell (SOFC) cathodes is crucial to improve their efficiency and thus to provide the basis for an economically feasible application of intermediate temperature SOFCs. In this work, minor amounts of Pt were doped into lanthanum strontium ferrite (LSF) thin film electrodes to modulate the material's oxygen exchange performance. Surprisingly, Pt was found to be incorporated on the B-site of the perovskite electrode as non metallic Pt 4+ . The polarization resistance of LSF thin film electrodes with and without additional Pt surface doping was compared directly after film growth employing in situ electrochemical impedance spectroscopy inside a PLD chamber ( i -PLD). This technique enables observation of the polarization resistance of pristine electrodes unaltered by degradation or any external contamination of the electrode surface. Moreover, growth of multi-layers of materials with different compositions on the very same single crystalline electrolyte substrate combined with in situ impedance measurements allow excellent comparability of different materials. Even a 5 nm layer of Pt doped LSF (1.5 at% Pt), i.e. a Pt loading of 80 ng cm -2 , improved the polarization resistance by a factor of about 2.5. In addition, p (O 2 ) and temperature dependent impedance measurements on both pure and Pt doped LSF were performed in situ and obtained similar activation energies and p (O 2 ) dependence of the polarization resistance, which allow us to make far reaching mechanistic conclusions indicating that Pt 4+ introduces additional active sites., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The oxygen exchange reaction mechanism on truly pristine surfaces of SOFC cathode materials (La 0.6 Sr 0.4 CoO 3- δ = LSC, La 0.6 Sr 0.4 FeO 3- δ = LSF, (La 0.6 Sr 0.4 ) 0.98 Pt 0.02 FeO 3- δ = Pt:LSF, SrTi 0.3 Fe 0.7 O 3- δ = STF, Pr 0.1 Ce 0.9 O 2- δ = PCO and La 0.6 Sr 0.4 MnO 3- δ = LSM) was investigated employing in situ impedance spectroscopy during pulsed laser deposition (i-PLD) over a wide temperature and p (O 2 ) range. Besides demonstrating the often astonishing catalytic capabilities of the materials, it is possible to discuss the oxygen exchange reaction mechanism based on experiments on clean surfaces unaltered by external degradation processes. All investigated materials with at least moderate ionic conductivity ( i.e. all except LSM) exhibit polarization resistances with very similar p (O 2 )- and T -dependences, mostly differing only in absolute value. In combination with non-equilibrium measurements under polarization and defect chemical model calculations, these results elucidate several aspects of the oxygen exchange reaction mechanism and refine the understanding of the role oxygen vacancies and electronic charge carriers play in the oxygen exchange reaction. It was found that a major part of the effective activation energy of the surface exchange reaction, which is observed during equilibrium measurements, originates from thermally activated charge carrier concentrations. Electrode polarization was therefore used to control defect concentrations and to extract concentration amended activation energies, which prove to be drastically different for oxygen incorporation and evolution (0.26 vs. 2.05 eV for LSF)., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

The interaction of light with solids has been of ever-growing interest for centuries, even more so since the quest for sustainable utilization and storage of solar energy became a major task for industry and research. With SrTiO 3 being a model material for an extensive exploration of the defect chemistry of mixed conducting perovskite oxides, it has also been a vanguard in advancing the understanding of the interaction between light and the electronic and ionic structure of solids. In the course of these efforts, many phenomena occurring during or subsequent to the illumination of SrTiO 3 have been investigated. Here, we give an overview of the numerous photoinduced effects in SrTiO 3 and their inherent connection to electronic structure and defect chemistry. In more detail, advances in the fields of photoconductivity, photoluminescence, photovoltages, photochromism and photocatalysis are summarized and their underlying elemental processes are discussed. In light of recent research, this review also emphasizes the fundamental differences between illuminating SrTiO 3 either at low temperatures (200 °C), where in addition to electronic processes, also photoionic interactions become relevant. A survey of the multitude of different processes shows that a profound and comprehensive understanding of the defect chemistry and its alteration under illumination is both vital to optimizing devices and to pushing the boundaries of research and advancing the fundamental understanding of solids., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

The interplay of structure, composition and electrical conductivity was investigated for Fe-doped SrTiO 3 thin films prepared by pulsed laser deposition. Structural information was obtained by reciprocal space mapping while solution-based inductively-coupled plasma optical emission spectroscopy and positron annihilation lifetime spectroscopy were employed to reveal the cation composition and the predominant point defects of the thin films, respectively. A severe cation non-stoichiometry with Sr vacancies was found in films deposited from stoichiometric targets. The across plane electrical conductivity of such epitaxial films was studied in the temperature range of 250-720 °C by impedance spectroscopy. This revealed a pseudo-intrinsic electronic conductivity despite the substantial Fe acceptor doping, i.e. conductivities being several orders of magnitude lower than expected. Variation of PLD deposition parameters causes some changes of the cation stoichiometry, but the films still have conductivities much lower than expected. Targets with significant Sr excess (in the range of several percent) were employed to improve the cation stoichiometry in the films. The use of 7% Sr-excess targets resulted in near-stoichiometric films with conductivities close to the stoichiometric bulk counterpart. The measurements show that a fine-tuning of the film stoichiometry is required in order to obtain acceptor doped SrTiO 3 thin films with bulk-like properties. One can conclude that, although reciprocal space maps give a first hint whether or not cation non-stoichiometry is present, conductivity measurements are more appropriate for assessing SrTiO 3 film quality in terms of cation stoichiometry., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Cubic Li 7 La 3 Zr 2 O 12 (LLZO) garnets are among the most promising solid electrolytes for solid-state batteries with the potential to exceed conventional battery concepts in terms of energy density and safety. The electrochemical stability of LLZO is crucial for its application, however, controversial reports in the literature show that it is still an unsettled matter. Here, we investigate the electrochemical stability of LLZO single crystals by applying electric field stress via macro- and microscopic ionically blocking Au electrodes in ambient air. Induced material changes are subsequently probed using various locally resolved analysis techniques, including microelectrode electrochemical impedance spectroscopy (EIS), laser induced breakdown spectroscopy (LIBS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), and microfocus X-ray diffraction (XRD). Our experiments indicate that LLZO decomposes at 4.1-4.3 V vs. Li + /Li, leading to the formation of Li-poor phases like La 2 Zr 2 O 7 beneath the positively polarized electrode. The reaction is still on-going even after several days of polarization, indicating that no blocking interfacial layer is formed. The decomposition can be observed at elevated as well as room temperature and suggests that LLZO is truly not compatible with high voltage cathode materials., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Filamentous fungi are well-established production hosts that feature a strong interconnection between morphology, physiology, and productivity. For penicillin production in Penicillium chrysogenum, industrial processes frequently favor a pellet morphology comprising compact hyphal agglomerates. Inherently these tightly packed entanglements lead to inactive, degrading sections within the pellet's core because of limitations. Optimal process design requires detailed knowledge of the nature of the limitations and localization of productive zones in the biomass, which is generally obtainable through modeling and complex analytical methods such as oxygen microelectrode and histological investigations. Methods that combine physiological and morphological insight are crucial yet scarce for filamentous fungi. In this study, we used time-of-flight secondary ion mass spectrometry in combination with oxygen and glucose tracer substrates, requiring little effort for sample preparation and measurement. Our method is capable of analyzing oxygen and substrate uptake in various morphological structures by the use of 18 O as a tracer. In parallel, we can assess productive biomass regions through identification of penicillin mass fragments to simultaneously study oxygen diffusion, substrate incorporation, and productive biomass sections.

The further development of solid oxide fuel and electrolysis cells (SOFC/SOEC) strongly relies on research activities dealing with electrode materials. Recent studies showed that under operating conditions many perovskite-type oxide electrodes are prone to changes of their surface composition, leading to severe changes of their electrochemical performance. This results in a large scatter of data in literature and complicates comparison of materials. Moreover, little information is available on the potentially excellent properties of surfaces immediately after preparation, that is, before any degradation by exposure to other gas compositions or temperature changes. Here, we introduce in situ impedance spectroscopy during pulsed laser deposition (IPLD) as a new method for electrochemical analysis of mixed ionic and electronic conducting (MIEC) thin films during growth. First, this approach can truly reveal the properties of as-prepared MIEC electrode materials, since it avoids any alterations of their surface between preparation and investigation. Second, the measurements during growth give information on the thickness dependence of film properties. This technique is applied to La 0.6 Sr 0.4 CoO 3-δ (LSC), one of the most promising SOFC/SOEC oxygen electrode material. From the earliest stages of LSC film deposition on yttria-stabilized zirconia (YSZ) to a fully grown thin film of 100 nm thickness, data are gained on the oxygen exchange kinetics and the defect chemistry of LSC. A remarkable reproducibility is found in repeated film growth experiments, not only for the bulk related chemical capacitance but also for the surface related polarization resistance (±10%). Polarization resistances of as-prepared LSC films are extraordinarily low (2.0 Ω cm 2 in 40 μbar O 2 at 600 °C). LSC films on YSZ and on La 0.95 Sr 0.05 Ga 0.95 Mg 0.05 O 3-δ (LSGM) single crystals exhibit significantly different electrochemical properties, possibly associated with the tensile strain of LSC on LSGM., Competing Interests: The authors declare no competing financial interest.

Perovskite oxide surfaces catalyze oxygen exchange reactions that are crucial for fuel cells, electrolyzers, and thermochemical fuel synthesis. Here, by bridging the gap between surface analysis with atomic resolution and oxygen exchange kinetics measurements, we demonstrate how the exact surface atomic structure can determine the reactivity for oxygen exchange reactions on a model perovskite oxide. Two precisely controlled surface reconstructions with (4 × 1) and (2 × 5) symmetry on 0.5 wt.% Nb-doped SrTiO 3 (110) were subjected to isotopically labeled oxygen exchange at 450 °C. The oxygen incorporation rate is three times higher on the (4 × 1) surface phase compared to the (2 × 5). Common models of surface reactivity based on the availability of oxygen vacancies or on the ease of electron transfer cannot account for this difference. We propose a structure-driven oxygen exchange mechanism, relying on the flexibility of the surface coordination polyhedra that transform upon dissociation of oxygen molecules.

Owing to its extraordinary high activity for catalysing the oxygen exchange reaction, strontium doped LaCoO 3 (LSC) is one of the most promising materials for solid oxide fuel cell (SOFC) cathodes. However, under SOFC operating conditions this material suffers from performance degradation. This loss of electrochemical activity has been extensively studied in the past and an accumulation of strontium at the LSC surface has been shown to be responsible for most of the degradation effects. The present study sheds further light onto LSC surface changes also occurring under SOFC operating conditions. In-situ near ambient pressure X-ray photoelectron spectroscopy measurements were conducted at temperatures between 400 and 790 °C. Simultaneously, electrochemical impedance measurements were performed to characterise the catalytic activity of the LSC electrode surface for O 2 reduction. This combination allowed a correlation of the loss in electro-catalytic activity with the appearance of an additional La-containing Sr-oxide species at the LSC surface. This additional Sr-oxide species preferentially covers electrochemically active Co sites at the surface, and thus very effectively decreases the oxygen exchange performance of LSC. Formation of precipitates, in contrast, was found to play a less important role for the electrochemical degradation of LSC.

Any substantial move of energy sources from fossil fuels to renewable resources requires large scale storage of excess energy, for example, via power to fuel processes. In this respect electrochemical reduction of CO 2 may become very important, since it offers a method of sustainable CO production, which is a crucial prerequisite for synthesis of sustainable fuels. Carbon dioxide reduction in solid oxide electrolysis cells (SOECs) is particularly promising owing to the high operating temperature, which leads to both improved thermodynamics and fast kinetics. Additionally, compared to purely chemical CO formation on oxide catalysts, SOECs have the outstanding advantage that the catalytically active oxygen vacancies are continuously formed at the counter electrode, and move to the working electrode where they reactivate the oxide surface without the need of a preceding chemical (e.g., by H 2 ) or thermal reduction step. In the present work, the surface chemistry of (La,Sr)FeO 3-δ and (La,Sr)CrO 3-δ based perovskite-type electrodes was studied during electrochemical CO 2 reduction by means of near-ambient pressure X-ray photoelectron spectroscopy (NAP-XPS) at SOEC operating temperatures. These measurements revealed the formation of a carbonate intermediate, which develops on the oxide surface only upon cathodic polarization (i.e., under sufficiently reducing conditions). The amount of this adsorbate increases with increasing oxygen vacancy concentration of the electrode material, thus suggesting vacant oxygen lattice sites as the predominant adsorption sites for carbon dioxide. The correlation of carbonate coverage and cathodic polarization indicates that an electron transfer is required to form the carbonate and thus to activate CO 2 on the oxide surface. The results also suggest that acceptor doped oxides with high electron concentration and high oxygen vacancy concentration may be particularly suited for CO 2 reduction. In contrast to water splitting, the CO 2 electrolysis reaction was not significantly affected by metallic particles, which were exsolved from the perovskite electrodes upon cathodic polarization. Carbon formation on the electrode surface was only observed under very strong cathodic conditions, and the carbon could be easily removed by retracting the applied voltage without damaging the electrode, which is particularly promising from an application point of view.

A single crystalline SrTiO 3 working electrode in a zirconia-based solid oxide electrochemical cell is illuminated by UV light at temperatures of 360-460 °C. In addition to photovoltaic effects, this leads to the build-up of a battery-type voltage up to more than 300 mV. After switching off UV light, this voltage only slowly decays. It is caused by UV-induced oxygen incorporation into the mixed conducting working electrode and thus by changes of the oxygen stoichiometry δ in SrTiO 3- δ under UV illumination. These changes of the oxygen content could be followed in time-dependent voltage measurements and also manifest themselves in time-dependent resistance changes during and after UV illumination. Discharge currents measured after UV illumination reveal that a large fraction of the existing oxygen vacancies in SrTiO 3 become filled under UV light. Additional measurements on cells with TiO 2 thin film electrodes show the broader applicability of this novel approach for transforming light into chemical energy and thus the feasibility of solid oxide photoelectrochemical cells (SOPECs) in general and of a "light-charged oxygen battery" in particular.

A study on charge transport properties of thin film Fe-doped SrTiO 3 epitaxially grown on Nb-doped SrTiO 3 is reported. Electric measurements between 350 °C and 750 °C show a transition from predominant ionic to electronic conduction and lower conductivity of the thin films compared to the bulk of polycrystalline samples. Defect chemical changes at elevated temperature were investigated by applying a bias voltage. A model is described which successfully predicts additional features such as inductive loops or extra semicircles measureable by impedance spectroscopy as well as the complicated time dependence of electric DC-measurements. With this model it is also possible to calculate the negligibly small ionic conductivity next to the dominating electronic conductivity in the high temperature regime. The ionic conductivity is referenced by oxygen isotope depth profiling. Changes of resistive states in Fe-doped SrTiO 3 thin films at high temperature and moderate fields are compared to room temperature resistive switching phenomena at high electric fields. A conductive filament based switching process is observed at room temperature, and the capability for forming such filaments and their electric properties is further analysed using microelectrodes.

We employed operando anomalous surface X-ray diffraction to investigate the buried interface between the cathode and the electrolyte of a model solid oxide fuel cell with atomic resolution. The cell was studied under different oxygen pressures at elevated temperatures and polarizations by external potential control. Making use of anomalous X-ray diffraction effects at the Y and Zr K-edges allowed us to resolve the interfacial structure and chemical composition of a (100)-oriented, 9.5 mol % yttria-stabilized zirconia (YSZ) single crystal electrolyte below a La 0.6 Sr 0.4 CoO 3-δ (LSC) electrode. We observe yttrium segregation toward the YSZ/LSC electrolyte/electrode interface under reducing conditions. Under oxidizing conditions, the interface becomes Y depleted. The yttrium segregation is corroborated by an enhanced outward relaxation of the YSZ interfacial metal ion layer. At the same time, an increase in point defect concentration in the electrolyte at the interface was observed, as evidenced by reduced YSZ crystallographic site occupancies for the cations as well as the oxygen ions. Such changes in composition are expected to strongly influence the oxygen ion transport through this interface which plays an important role for the performance of solid oxide fuel cells. The structure of the interface is compared to the bare YSZ(100) surface structure near the microelectrode under identical conditions and to the structure of the YSZ(100) surface prepared under ultrahigh vacuum conditions.

Resistive switches based on anionic electronic conducting oxides are promising devices to replace transistor-based memories due to their excellent scalability and low power consumption. In this study, we create a model switching system by manufacturing resistive switches based on ultrathin 5 nm, epitaxial, and grain boundary-free strontium titanate thin films with subnanometer surface roughness. For our model devices, we unveil two competing nonvolatile resistive switching processes being of different polarities: one switching in clockwise and the other in counterclockwise direction. They can be activated selectively with respect to the effective switching voltage and time applied to the device. Combined analysis of both processes with electrical DC-methods and electrochemical impedance spectroscopy reveals that the first resistive switching process is filament-based and exhibits counterclockwise bipolar resistive switching. The R(OFF)/R(ON) resistance ratio of this process is extremely stable and can be tuned in the range 5-25 depending on the switching voltage and time. Excitingly, at high electric field strength a second bipolar resistive switching process was found. This process is clockwise and, therefore, reveals the opposite polarity switching direction when compared to the first one. Both processes do not obstruct each other, consequently, stable 1, 2, or even 3 crossover current-voltage (I-V) characteristics can be addressed for the memory bits. Equivalent circuit model analysis and fitting of impedance characteristics unequivocally show for the created grain boundary free switches that the oxide's defects and its carrier distribution close to the electrode interface contribute to the resistive switching mechanism. The addressability of two sets of resistive ON and OFF states in one device through electric field strength and switching time offers exciting new operation schemes for memory devices.

In this paper, we present a strategy to use interfacial strain in multilayer heterostructures to tune their resistive response and ionic transport as active component in an oxide-based multilayer microdot device on chip. For this, fabrication of strained multilayer microdot devices with sideways attached electrodes is reported with the material system Gd0.1Ce0.9O(2-δ)/Er2O3. The fast ionic conducting Gd0.1Ce0.9O(2-δ) single layers are altered in lattice strain by the electrically insulating erbia phases of a microdot. The strain activated volume of the Gd0.1Ce0.9O(2-δ) is investigated by changing the number of individual layers from 1 to 60 while keeping the microdot at a constant thickness; i.e., the proportion of strained volume was systematically varied. Electrical measurements showed that the activation energy of the devices could be altered by Δ0.31 eV by changing the compressive strain of a microdot ceria-based phase by more than 1.16%. The electrical conductivity data is analyzed and interpreted with a strain volume model and defect thermodynamics. Additionally, an equivalent circuit model is presented for sideways contacted multilayer microdots. We give a proof-of-concept for microdot contacting to capture real strain-ionic transport effects and reveal that for classic top-electrode contacting the effect is nil, highlighting the need for sideways electric contacting on a nanoscopic scale. The near order ionic transport interaction is supported by Raman spectroscopy measurements. These were conducted and analyzed together with fully relaxed single thin film samples. Strain states are described relative to the strain activated volumes of Gd0.1Ce0.9O(2-δ) in the microdot multilayer. These findings reveal that strain engineering in microfabricated devices allows altering the ionic conduction over a wide range beyond classic doping strategies for single films. The reported fabrication route and concept of strained multilayer microdots is a promising path for applying strained multilayer oxides as active new building blocks relevant for a broad range of microelectrochemical devices, e.g., resistive switching memory prototypes, resistive or electrochemical sensors, or as active catalytic solid state surface components for microfuel cells or all-solid-state batteries.

The oxygen exchange and diffusion properties of La 0.6 Sr 0.4 CoO 3 - δ thin films on yttria stabilized zirconia were analyzed by impedance spectroscopy and 18 O tracer experiments. The investigations were performed on the same thin film samples and at the same temperature (400 °C) in order to get complementary information by the two methods. Electrochemical impedance spectroscopy can reveal resistive and capacitive contributions of such systems, but an exact interpretation of the spectra of complex oxide electrodes is often difficult from impedance data alone. It is shown that additional isotope exchange depth profiling can significantly help interpreting impedance spectra by giving reliable information on the individual contribution and exact location of resistances (surface, electrode bulk, interface). The measurements also allowed quantitative comparison of electrode polarization resistances obtained by different methods.

Cation diffusion was investigated in La0.6Sr0.4CoO3-δ (LSC) thin films on (100) yttria stabilized zirconia in the temperature range 625-800 °C. Isotopic ((86)Sr) and elemental tracers (Fe, Sm) were used to establish diffusion profiles of the cations in bi- and multi-layered thin films. The profiles were analyzed by time of flight-secondary ion mass spectrometry (ToF-SIMS). Grain and grain boundary diffusion coefficients of the cations were determined for LSC thin films with columnar grains - diffusion along grain boundaries is shown to be about three orders of magnitude faster than in grains. This could be verified for thin films with different grain size. A- and B-site cations showed very similar temperature dependencies with activation energies of ∼3.5 eV for bulk and ∼4.1 eV for grain boundary diffusion. The importance of cation diffusivities for surface segregation of Sr and thus for a major degradation mechanism of LSC cathodes in solid oxide fuel cells is discussed.

A novel operation mode for time of flight-secondary ion mass spectrometry (ToF-SIMS) is described for a TOF.SIMS 5 instrument with a Bi-ion gun. It features sub 100 nm lateral resolution, adjustable primary ion currents and the possibility to measure with high lateral resolution as well as high mass resolution. The adjustment and performance of the novel operation mode are described and compared to established ToF-SIMS operation modes. Several examples of application featuring novel scientific results show the capabilities of the operation mode in terms of lateral resolution, accuracy of isotope analysis of oxygen, and combination of high lateral and mass resolution. The relationship between high lateral resolution and operation of SIMS in static mode is discussed.

The influence of lattice strain on the oxygen exchange kinetics and diffusion in oxides was investigated on (100) epitaxial La1-xSrxCoO3-δ (LSC) thin films grown by pulsed laser deposition. Planar tensile and compressively strained LSC films were obtained on single-crystalline SrTiO3 and LaAlO3. 18O isotope exchange depth profiling with ToF-SIMS was employed to simultaneously measure the tracer surface exchange coefficient k* and the tracer diffusion coefficient D* in the temperature range 280-475 °C. In accordance with recent theoretical findings, much faster surface exchange (∼4 times) and diffusion (∼10 times) were observed for the tensile strained films compared to the compressively strained films in the entire temperature range. The same strain effect--tensile strain leading to higher k* and D*--was found for different LSC compositions (x=0.2 and x=0.4) and for surface-etched films. The temperature dependence of k* and D* is discussed with respect to the contributions of strain states, formation enthalpy of oxygen vacancies, and vacancy mobility at different temperatures. Our findings point toward the control of oxygen surface exchange and diffusion kinetics by means of lattice strain in existing mixed conducting oxides for energy conversion applications.

Thrombotic occlusion of an epicardial coronary artery on the grounds of atherosclerotic plaque is considered the ultimate step in AMI (acute myocardial infarction). However, the precise pathophysiological mechanisms underlying acute coronary occlusion are not fully understood. We have analysed proteomic profiles of systemic plasma and plasma derived from the site of coronary plaque rupture of non-diabetic patients with STEMI (ST-segment elevation myocardial infarction). Label-free quantification of MS/MS (tandem MS) data revealed differential regulation of complement cascade components and a decrease in anti-thrombotic PEDF (pigment epithelium-derived factor) between CS (culprit site)-derived plasma and systemic plasma. PEDF, which is known to have a protective role in atherothrombosis, was relatively decreased at the CS, with a level of expression inverse to local MMP-9 (matrix metalloproteinase-9) activity. CS plasma displayed enhanced proteolytic activity towards PEDF. Proteomics of coronary thrombus aspirates indicate that PEDF processing is associated with coronary plaque rupture.

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.

The oxygen exchange kinetics of platinum on yttria-stabilized zirconia (YSZ) was investigated by means of geometrically well-defined Pt microelectrodes. By variation of electrode size and temperature it was possible to separate two temperature regimes with different geometry dependencies of the polarization resistance. At higher temperatures (550-700 °C) an elementary step located close to the three phase boundary (TPB) with an activation energy of ∼1.6 eV was identified as rate limiting. At lower temperatures (300-400 °C) the rate limiting elementary step is related to the electrode area and exhibited a very low activation energy in the order of 0.2 eV. From these observations two parallel pathways for electrochemical oxygen exchange are concluded.The nature of these two elementary steps is discussed in terms of equivalent circuits. Two combinations of parallel rate limiting reaction steps are found to explain the observed geometry dependencies: (i) Diffusion through an impurity phase at the TPB in parallel to diffusion of oxygen through platinum - most likely along Pt grain boundaries - as area-related process. (ii) Co-limitation of oxygen diffusion along the Pt|YSZ interface and charge transfer at the interface with a short decay length of the corresponding transmission line (as TPB-related process) in parallel to oxygen diffusion through platinum.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Atherosclerotic plaque rupture with subsequent mural thrombus formation is considered the main event compromising epicardial flow in acute myocardial infarction (AMI). However, the precise mechanisms underlying acute coronary occlusion are unknown. We compared the proteomic profiles of systemic plasma and plasma derived from the site of thrombus formation of patients with AMI by two-dimensional gel electrophoresis and ELISA. We identified a local activation of the complement system, with selective accumulation of the complement activator C-reactive protein (CRP) and the downstream complement effectors C3a and C5a. CRP in coronary thrombus co-localised with C1q and C3 immunoreactivities, suggesting classical complement activation. In vitro, coronary thrombus derived plasma enhanced neutrophil chemotaxis in a C5a dependent fashion. In vivo, neutrophil accumulation at the site of thrombus formation paralleled the time delay from symptom onset to first balloon inflation or aspiration, and was correlated with C5a and enzymatic infarct size. We present the first direct evidence for localised complement activation in acute coronary thrombi. Our data indicate that local complement effectors amplify the vascular occlusion process in AMI by enhanced neutrophil recruitment.

Interpretation of proteome data with a focus on biomarker discovery largely relies on comparative proteome analyses. Here, we introduce a database-assisted interpretation strategy based on proteome profiles of primary cells. Both 2-D-PAGE and shotgun proteomics are applied. We obtain high data concordance with these two different techniques. When applying mass analysis of tryptic spot digests from 2-D gels of cytoplasmic fractions, we typically identify several hundred proteins. Using the same protein fractions, we usually identify more than thousand proteins by shotgun proteomics. The data consistency obtained when comparing these independent data sets exceeds 99% of the proteins identified in the 2-D gels. Many characteristic differences in protein expression of different cells can thus be independently confirmed. Our self-designed SQL database (CPL/MUW - database of the Clinical Proteomics Laboratories at the Medical University of Vienna accessible via www.meduniwien.ac.at/proteomics/database) facilitates (i) quality management of protein identification data, which are based on MS, (ii) the detection of cell type-specific proteins and (iii) of molecular signatures of specific functional cell states. Here, we demonstrate, how the interpretation of proteome profiles obtained from human liver tissue and hepatocellular carcinoma tissue is assisted by the Clinical Proteomics Laboratories at the Medical University of Vienna-database. Therefore, we suggest that the use of reference experiments supported by a tailored database may substantially facilitate data interpretation of proteome profiling experiments.

The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of protein concentrations presents problems with quantitation. The extreme complexity of the proteome in body fluids presents enormous challenges and requires the establishment of standard operating procedures for handling of specimens, increasing sensitivity for detection and bioinformatical tools for distribution of proteomic data into the public domain. From studies of in vitro diagnostics, especially in clinical chemistry, it is evident that most errors occur in the preanalytical phase and during implementation of the diagnostic strategy. This is also true for clinical proteomics, and especially for fluid proteomics because of the multiple pretreatment processes. These processes include depletion of high-abundance proteins from plasma or enrichment processes for urine where biological variation or differences in proteolytic activities in the sample along with preanalytical variables such as inter- and intra-assay variability will likely influence the results of proteomics studies. However, before proteomic analysis can be introduced at a broader level into the clinical setting, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement and data analysis needs to be improved. In this review, we discuss the recent technological advances and applications that fulfil the criteria for clinical proteomics, with the focus on fluid proteomics. These advances relate to preanalytical factors, analytical standardization and quality-control measures required for effective implementation into routine laboratory testing in order to generate clinically useful information. With new disease biomarker candidates, it will be crucial to design and perform clinical studies that can identify novel diagnostic strategies based on these techniques, and to validate their impact on clinical decision-making.

The aim of this study is to quantify the effectivity of the depletion of human high-abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2-DE and bottom-up shotgun MS combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high-abundance protein depletion. Nano-liquid chromatography linked MS allowed the efficient identification of several low-abundant proteins that were not identified on the 2-DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.

Objective: Mounting evidence suggests that activation of complement, an important constituent of innate immunity, contributes to atherosclerosis. Here we investigated the expression of complement components (CCs) in the setting of experimental and clinical hypercholesterolemia, a major risk factor for atherosclerosis, their effects on vascular smooth muscle cell (VSMC) and macrophage proliferation, and the underlying molecular mechanisms., Methods: For this study we analyzed the mRNA and protein expression of several CCs in plasma and aorta of hypercholesterolemic atherosclerosis-prone apolipoprotein E-null mice (apoE-KO) and in plasma of normocholesterolemic subjects and familial hypercholesterolemia (FH) patients. We also carried out in vitro molecular studies to assess the role of CCs on the control of macrophage and VSMC proliferation., Results: Fat-fed apoE-KO mice experiencing severe hypercholesterolemia (approximately 400 mg/dL), but not fat-fed wild-type controls with plasma cholesterol level<110 mg/dL, displayed in aortic tissue upregulation of several CC mRNAs, including C3, C4, C1s, and C1q. In apoE-KO mice, induction of C3 mRNA was already apparent two days after fat feeding when hypercholesterolemia was manifested yet atherosclerotic lesions were absent or incipient. Rapid C3 and C4 protein upregulation was also observed in the plasma of fat-fed apoE-KO mice, and FH patients exhibited higher plasmatic C3a, C4 gamma chain, C1s and C3c alpha chain protein levels than normocholesterolemic subjects. In vitro, C3 and C3a, but not C3a-desArg, C4 and C1q, promoted macrophage and VSMC proliferation through Gi protein-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2). We also found that C3-enriched FH plasma evoked a stronger mitogenic response in macrophages than normocholesterolemic plasma, and treatment with anti-C3 antibodies eliminated this difference., Conclusions: Both experimental and clinical hypercholesterolemia coincides with a concerted activation of several CCs. However, only C3 and C3a elicited a mitogenic response in cultured VSMCs and macrophages through Gi protein-dependent ERK1/2 activation. Thus, excess of C3/C3a in hypercholesterolemic apoE-KO mice and FH patients may contribute to atheroma growth by promoting neointimal cell proliferation.

Proteome profiling techniques rely on the separation of proteins or peptides and their subsequent quantification. The reliability of this technique is still limited because a proteome profiling result does not necessarily represent the true protein composition of the analysed sample, thus seriously hampering proper data interpretation. Many experimentally observed proteome alterations are biologically not significant. It was the aim of this study to use the knowledge of the biological context of proteins in order to establish optimised proteome profiling protocols. While 2-D spot patterns of total cell protein fractions were found to poorly represent the true protein composition, purified subcellular protein fractions were found to better represent the protein composition of the analysed sample. The application of a standardised protocol to different kinds of cells revealed several striking observations. Firstly, the protein composition of cultured cells of various origins is very similar. Secondly, proteome alterations observed with the described protocols do make sense from a biologic point of view and may thus be considered as truly representative for the analysed samples. Thirdly, primary white blood cells isolated from different donors were found to show minor, but reproducible and significant individual differences. We designate the consideration of known properties of identified proteins in proteome profiles as a knowledge-based approach. The present data suggest that this approach may tremendously help to improve the applied techniques and assess the results. We demonstrate that the fulfilment of well-defined criteria of proteome profiles eventually results in reliable and biologically relevant data.

AP-1 (Activating Protein 1) transcription factor activity is tightly regulated at multiple levels, including dimer formation (i.e., Fos/Jun). Here we show that the intermediate filament protein lamin A/C suppresses AP-1 function through direct interaction with c-Fos, and that both proteins can interact and colocalize at the nuclear envelope (NE) in mammalian cells. Perinuclear localization of c-Fos is absent in Lmna-null cells but can be restored by lamin A overexpression. In vitro, preincubation of c-Fos with lamin A prior to the addition of c-Jun inhibits AP-1 DNA-binding activity. In vivo, overexpression of lamin A reduces the formation of c-Fos/c-Jun heterodimers, and suppresses AP-1 DNA-binding and transcriptional activity. Notably, c-Fos colocalizes with lamin A/C at the NE in starvation-synchronized quiescent cells lacking detectable AP-1 DNA binding. In contrast, serum-induced AP-1 DNA-binding activity coincides with abundant nucleoplasmic c-Fos expression without changes in lamin A/C localization. We also found that Lmna-null cells display enhanced proliferation. In contrast, lamin A overexpression causes growth arrest, and ectopic c-Fos partially overcomes lamin A/C-induced cell cycle alterations. We propose lamin A/C-mediated c-Fos sequestration at the NE as a novel mechanism of transcriptional and cell cycle control.

Mitogenic stimulation of Raf-1 is a complex yet incompletely understood process involving membrane relocalization and phosphorylation of activating residues. We recently reported that Raf-1-associated protein phosphatase 2A contributes to kinase activation, an effect mediated via Ser-259 of Raf-1. Here, we show that mitogens stimulate Ser-259 dephosphorylation and Raf-1/protein phosphatase 2A association concomitantly with membrane accumulation and activation of Raf-1. Blocking Ser-259 dephosphorylation inhibits the two latter events, but it does not prevent activation of a S259A Raf-1 mutant, which is preferentially localized at the membrane independently of mitogenic stimulation. Inhibition of Ser-259 dephosphorylation has no effect on the activation of membrane-tethered Raf-1 (Raf-1CAAX). These data show that Ser-259 dephosphorylation contributes to Raf-1 activation by supporting its membrane accumulation rather than by increasing the specific activity of the kinase and provide a mechanistic basis for the support of kinase activation by Raf-1-associated protein phosphatase 2A.

The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.

We isolated two proteins, ECP-51 and ECP-54, from human erythrocyte cytosol by affinity chromatography using a peptide of the integral membrane protein stomatin as bait. Partial amino acid sequence information obtained by microsequencing allowed us to clone the respective cDNAs. Analysis of the nucleotide sequences revealed that ECP-51 and ECP-54 are homologous (44.2% amino acid identity) and contain ATP-binding sites. ECP-54 was identified as TIP49/RUVBL1/NMP238, which is a component of a large nuclear protein complex, possibly the RNA polymerase II holoenzyme; ECP-51 is a novel protein. Using the two-hybrid system, we showed that these proteins interact with each other. The interaction of ECP-51 and ECP-54 with the stomatin peptide and the localization to the nucleus and cytoplasm suggest an additional function for these proteins as chaperone components.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

The solubilization of plasma membrane receptors through proteolytic cleavage of the ligand binding domain at the cell surface is an important mechanism for regulating cytokine function and receptor signaling. The inhibition of the shedding of a variety of receptors by synthetic inhibitors of the matrix metalloproteinases (MMPs) implicates metalloproteinases in this regulatory event. We examined the effects of two naturally occurring tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor alpha receptor type I (TNFalpha-RI; Mr 55,000) and TNFalpha-RII (Mr 75,000) by the Colo 205 human colon adenocarcinoma cell line. Culture of Colo 205 cells for 48 h resulted in the shedding of both TNFalpha-RI and TNFalpha-RII, as determined by ELISA. The shedding of TNFalpha receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibited in a dose-dependent manner by the following synthetic MMPIs: batimastat and marimastat (BB-94 and BB-2516, respectively, British Biotech, Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceuticals); and RO31-9790 (Roche), with IC50s ranging from 3.2 to 38.0 microM. Similarly, TIMP-2 from two different sources reproducibly inhibited the shedding of both TNFalpha-RI and TNFalpha-RII in a dose-dependent manner (IC50 = 286 +/- 33 nM for TNFalpha-RI shedding and 462 +/- 52 nM for shedding of TNFalpha-RII). The inhibition of TNFalpha-RI shedding was confirmed in the SW626 human ovarian adenocarcinoma cell line. The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNFalpha receptors retained on the surface of Colo 205 cells, as determined by flow cytometry. Inhibition of TNFalpha receptor shedding with TIMP-2 occurs at molar concentrations 10-100 times less than those required with low molecular weight, synthetic MMPIs but at concentrations greater than those required to inhibit collagen degradation. Modulation of TNFalpha receptor shedding by TIMP-2 could have important implications for the pleiotropic effects of TNFalpha in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.