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Abstract The application of hydrogen proton exchange membrane fuel cells (PEMFC) in greenhouse gas emission free heavy-duty vehicles requires extremely durable PEMFC components with service lives in the range of 30,000 h. Hence suitable test and analysis methods are required that reflect realistic operation scenarios, but significantly accelerate aging. For this purpose, a dynamic accelerated stress test was developed, which is coupled with a comprehensive in-depth in-situ and ex-situ analysis program to determine the aging processes of a PEMFC membrane electrode assembly (MEA). The test comprehends dynamic cycling between low, moderate and high load, different temperature and humidity conditions as well as recovery sequences to distinguish between reversible and irreversible failure modes. All phases of the PEMFC system (i.e. solid, liquid and gaseous) are monitored on-line during aging by sophisticated electrochemical, mass spectrometric and ion chromatographic analytical methods. The structural and elemental composition of the MEA before and after the aging program (post-mortem) are investigated by X-ray fluorescence, scanning and transmission electron microscopy. This program was able to age a commercial PEMFC to end-of-life in 1000 h, while providing an accurate picture of the aging processes involved.

Background: The insulin receptor-related receptor (IRR) was originally discovered due to its high homology to the other family members (insulin receptor and insulin-like growth factor 1 receptor). We determined that IRR can be activated by mildly alkaline extracellular media and has typical features of the ligand-receptor interaction, including its specificity and dose-dependence. Since pH-sensitive properties of IRR are determined by its ectodomain; therefore, we chose as an option to study the soluble extracellular domain IRR. Methods: The investigation carried out in Titan Krios 60-300 TEM/STEM (FEI, USA) CryoEM, equipped with direct electron detector Falcon II (FEI, USA) and Cs image corrector (CEOS, Germany), at an accelerating voltage of 300 kV. Data processing and 3D reconstruction were carried out using computing resources of the Federal Collective Usage Center Complex for Simulation and Data Processing for Mega-Science Facilities at NRC “Kurchatov Institute.” Results: The obtained 2D classifications of particles of the ectodomain IRR at a neutral pH form several 3D models. This indicates that the ectodomain has several possible conformations, which is consistent with our previously obtained data using SEC-SAXS and AFM. In the future, additional careful data processing is required, as well as studies of the IRR ectodomain in mildly alkaline pH. Conclusion: In this study, we presented the structural characteristics of the IRR ectodomain obtained by CryoEM. These results are an important step towards understanding the mechanism of functioning of the IRR.

Background: It was shown that when exposed to extreme environment Dps-DNA co-crystallization takes place in Escherichia coli as a protective mechanism. However, to this date most of the proposed models of co-crystals were not backed up with any direct methods. Recent developments in Cryo Electron Tomography allow to visualize co-crystals with high spacial resolution in nearly native state. Methods: The investigations were carried out in a Titan Krios 60-300 TEM/STEM (FEI, USA) CryoEM, equipped with direct electron detector Falcon II (FEI, USA) and Cs image corrector (CEOS, Germany), at accelerating voltage of 300 kV. Tilt series were collected automatically using FEI Tomography software in low-dose mode which allowed to minimize the radiation damage. Subtomogram averaging was performed in Relion2 software package. Data processing and 3D reconstruction were carried out using computing resources of the Federal Collective Usage Center Complex for Simulation and Data Processing for Mega-Science Facilities at NRC “Kurchatov Institute”. Results: In this study, different types of Dps-DNA co-crystals were observed in vitro. Structural characteristics of the co-crystals were revealed for the first time by Cryo Electron Tomography. Subtomogram averaging allowed the direct visualization of Dps and DNA molecules in co-crystals. It was shown that the first type of co-crystals exhibits lamellar morphology and is formed by Dps layers alternating with DNA strands. The co-crystals adopt central symmetric triclinic crystal structure. The second type adopts body-centered cubic crystal lattice with mutually perpendicular DNA strands packed between Dps molecules. We assume that co-crystals’ structure could be dependent on the parameters of the utilized Dps and DNA solutions. Conclusion: In this study, we established structural characteristics of different types of Dps-DNA co-crystals. The obtained results are a vital step towards understanding the mechanism of protective Dps-DNA co-crystallization process.

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps−DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps−DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.