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Clay rocks are investigated as potential host rocks for high-level nuclear waste (HLW) repositories in many countries. The Sandy Facies of Opalinus Clay (SF-OPA), as accessible in the Mont Terri rock laboratory, exhibits a pronounced heterogeneity, which is as well expected for lower cretaceous clay rocks, that are among the potential host rocks for the German HLW repository.Aim of the DR-D experiment is to characterize the heterogeneity of SF-OPA on the m-cm scale via seismic tomography and borehole seismic characterization. Borehole logging and ex-situ drill core characterization provide information on rock heterogeneity on smaller scales.Within the area, characterized by seismic tomography, a radio-tracer diffusion experiment will be set up, with the aim to correlate the observed diffusion behavior with the heterogeneous rock structure.The first drilling campaign took place in May 2021. The seismic tomography survey of the experimental area was performed in October 2021. Processing and analysis of the seismic data, as well as drill core characterization are currently underway.In this contribution we present the general concept and layout of the DR-D experiment, as well as first results.We acknowledge funding by the German Federal Ministry of Education and Research (BMBF) (Grant 02NUK053A-E), the Helmholtz Association (Grant SO-093), BGR, and BGE. Special thanks to swisstopo and the Mont-Terri project for help, advice, and support.

Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.

The Federal Company for Radioactive Waste Disposal (BGE) is responsible for the search for a site with the best possible safety for the disposal of high-level radioactive waste in Germany. The site selection procedure is regulated in a law that was adopted by the German Federal Parliament (Repository Site Selection Act – StandAG, 2017, last updated 2020) and aims to be a participatory, transparent, learning, and self-questioning process based on scientific expertise. The first step of the first phase of the site selection procedure was completed in September 2020 and resulted in the identification of sub-areas that give reason to expect favorable geological conditions for the long-term storage of nuclear waste in the subsurface. These sub-areas cover approximately 54 % of Germany and are located in three different host rocks: rock salt – halite, claystone, and crystalline rock. The challenge for the next step is to find suitable siting regions within the previously determined sub-areas that are then considered further in the next phase of the site selection procedure. In the following, the methodology of the so-called representative preliminary safety analyses is described, which constitute one of the tools to identify siting regions, and some first insight on how they are planned to be implemented in practice is given.

The Federal Company for Radioactive Waste Disposal (BGE) is the German waste management organisation responsible for implementing the search for a site with the best possible safety for the disposal of high-level radioactive waste for at least 1 million years, following the amendments of the Repository Site Selection Act in 2017. The selection procedure is meant to be a participatory, transparent, learning and self-questioning process based on scientific expertise. It consists of three phases with an increasing level of detail. The first step of the first phase of the site selection procedure was completed in September 2020 and resulted in the identification of 90 subareas that give reason to expect favourable geological conditions for the safe disposal. The potentially suitable subareas cover approximately 54 % of Germany and are located in three different host rocks: rock salt (halite), claystone and crystalline rock. The second step of phase one is currently in progress and includes the so-called representative preliminary safety analyses that aim to assess the extent to which the safe containment of the radioactive waste can be expected. Representative preliminary safety analyses are one of the foundations for deciding whether an area will be considered for surface-based exploration in the next phase of the site selection procedure. Within the preliminary safety analyses, the behaviour of the disposal system is analysed in its entirety, across all operational phases of the repository and under consideration of possible future evolution of the disposal system with respect to the safe containment of the radioactive waste. The development of a database is described, which aims to systematically document and provide the framework needed for the analyses of the disposal systems in the subareas regarding the safe containment of the radionuclides over the assessment period of 1 million years. This database includes the vast amount of information about the different components of the disposal system. This includes also the geological setting, the technical conception of the repository and compilations of values for the physical, geoscientific, and technical parameters characterising the various barriers of the disposal system. Furthermore, a self-contained derivation of expected and deviating future evolution of the disposal system and its geological setting is included; following the so-called features, events and processes (FEP) strategy.

The Federal Company for Radioactive Waste Disposal mbH (BGE mbH) is as Germans waste management organization responsible to implement the search for a site with the best possible safety for the disposal of high-level radioactive waste for at least one million years, following the amendments of the Repository Site Selection Act in 2017. The selection procedure is meant to be a participatory, transparent, learning and self-questioning process based on scientific expertise.This contribution will provide an overview of the methodology of the forthcoming preliminary safety assessments as a major part of the next steps in the site selection procedure. This procedure overall consists of three phases with increasing level of detail for identification of the best site. The first phase consists of two steps. The objective of the first step was to determine sub-areas in the three considered host rocks, salt (halite), clay and crystalline rock, by applying legally defined exclusion criteria, minimum requirements and geoscientific weighing criteria. 90 sub-areas that cover approximately 54 % of the area of Germany were identified due to their general suitable geological conditions. The result was published in September 2020.The second step of phase one is currently in progress and consists of representative preliminary safety assessments that aim to assess the safety of the repository system as well as its robustness. The requirements for the preliminary safety assessments in the site selection procedure are defined by a governmental directive released in October 2020. Representative preliminary safety assessments have to be performed for each sub-area and consist of the compilation of all geoscientific information relevant to the safety of a repository, the development of preliminary safety and repository concepts and the analysis of the repository system. In addition, a systematically identification and characterization of uncertainties has to be undertaken and the need for exploration, research and development must be determined. The application of the representative preliminary safety assessments as well as the following renewed application of geoscientific weighing criteria will lead to the identification of siting regions within the larger sub-areas of step one. These regions will be considered, first for surface-based geoscientific and geophysical exploration, including i.e. seismic exploration and drilling of boreholes. Subsequently the last phase of the site selection will proceed with subsurface exploration. Finally, all suitable sites will be proposed and the German government will decide the actual site. This process is expected to be finalized in 2031.

According to the 'Act on the Organizational Restructuring in the Field of Radioactive Waste Disposal' the BGE was established in 2016. The amended 'Repository Site Selection Act' (StandAG) came into force in July 2017 and forms the base for the site selection by clearly defining the procedure. According to the StandAG the BGE implements the participative, science-based, transparent, self-questioning and learning procedure with the overarching aim to identify the site for a high-level radioactive waste (HLW) repository in a deep geological formation with best possible safety conditions for a period of one million years.The German site selection procedure consists of three phases, of which Phase 1 is divided into two steps. Starting with a blanc map of Germany, the BGE completed Step 1 in September 2020 and identified 90 individual sub-areas that provide favorable geological conditions for the safe disposal of HLW in the legally considered host rocks; rock salt, clay and crystalline rock. Based on the results of Step 1, the on-going Step 2 will narrow down these sub-areas to siting regions for surface exploration within Phase 2 (§ 14 StandAG). Central to the siting process are representative (Phase 1), evolved (Phase 2) and comprehensive (Phase 3) preliminary safety assessments according to § 27 StandAG.The ordinances on 'Safety Requirements' and 'Preliminary Safety Assessments' for the disposal of high-level radioactive waste from October 2020 regulate the implementation of the preliminary safety assessments within the different phases of the siting process. Section 2 of the 'Safety Requirements' ordinance provides requirements to evaluate the long-term safety of the repository system; amongst others, it states that all potential effects that may affect the long-term safety of the repository system need to be systematically identified, described and evaluated as “expected” or “divergent” evolutions. Additionally, the ordinance on 'Preliminary Safety Assessments' states in § 7, amongst others, that the geoscientific long-term prediction is a tool to identify and to evaluate geogenic processes and to infer “expected” and “divergent” evolutions from those. Hence, considering the time period of one million years for the safe disposal of the HLW and the legal requirements, it is essential to include long-term climate evolution in the German site selection process to evaluate the impact of various climate-related scenarios on the safety of the whole repository system.To better understand and evaluate the influence of climate-related processes on the long-term safety of a HLW repository, climate-related research will be a part of the BGE research agenda. Potential research needs may address i) processes occurring on glacial – interglacial timescales (e.g. the inception of the next glaciation, formation and depth of permafrost, glacial troughs, sub-glacial channels, sea-level rise, orbital forcing) and their future evolutions, ii) effects on the host rocks and the barrier system(s) as well as iii) the uncertainties related to these effects but also to general climate models and predictions.

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Forster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (kFRET), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (rET) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

In Germany, the site selection for a repository for radioactive waste in deep geological formations was (re-) started in 2017 with the Repository Site Selection Act coming into force. The Site Selection Act envisages preliminary safety assessments as a measure to ensure the safety of a considered site.The focus of the presentation will be the methodology of the preliminary safety assessments as it is derived from the legal requirements. In this context, the Federal Ministry for Environment, Nature Conservation and Nuclear Safety published the draft of the regulation on the safety requirements for the disposal of high-level radioactive waste in summer 2019. Article 2 of this regulation contains the requirements for the implementation of preliminary safety assessments in the site selection procedure. One essential aspect is the systematical identification and characterization of uncertainties. We will discuss the key features of the handling of uncertainties in the site selection procedure, especially with regard to numerical reactive transport modelling. The German Site Selection Act is divided into several steps with increasing level of detail. The identification and quantification of uncertainties plays a major role to improve quality and plausibility in each step. Well-prepared explorations for instance, need to be addressed in a way to minimise data uncertainties. In addition, the handling of uncertainties in safety assessments on an international level is evaluated.

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells. In vitro, miuraenamide A acts as an actin nucleating, F-actin polymerizing and stabilizing compound, just like described for jasplakinolide. However, in contrast to jasplakinolide, miuraenamide A competes with cofilin, but not gelsolin or Arp2/3 for binding to F-actin. We propose a binding mode of miuraenamide A, explaining both its similarities and its differences to jasplakinolide. Molecular dynamics simulations suggest that the bromophenol group of miurenamide A interacts with residues Tyr133, Tyr143, and Phe352 of actin. This shifts the D-loop of the neighboring actin, creating tighter packing of the monomers, and occluding the binding site of cofilin. Since relatively small changes in the molecular structure give rise to this selectivity, actin binding compounds surprisingly are promising scaffolds for creating actin binders with specific functionality instead of just “stabilizers”.

Actin filament dynamics underlie key cellular processes, such as cell motility. Although actin filament elongation has been extensively studied under the past decades, the mechanism of filament nucleation remains unclear. Here, we immobilized gelsolin, a pointed-end nucleator, at the bottom of zero-mode waveguides to directly monitor the early steps of filament assembly. Our data revealed extensive dynamics and that only one, of two populations, elongates. Annalysis of the kinetics revealed a more stable trimer but a less stable tetramer in the elongating population compared to the non-elongating one. Furthermore, blocking flattening, the conformational change associated with filament formation, prevented the formation of both types of assemblies. Thus, flattening and the initial monomer arrangement determine gelsolin-mediated filament initiation.

The key drivers controlling the redox state of seawater and sediment pore waters in low energy environments can be inferred from redox-sensitive trace elements (RSTE), molecular biomarkers and trace metal isotopes. Here, we apply a combination of these tools to the Upper Permian Kupferschiefer (T1) from the Thuringian Basin, deposited in the southern part of the semi-enclosed Kupferschiefer Sea. Enrichment patterns of the RSTEs molybdenum (Mo) and uranium (U) as well as biomarker data attest to the rapid development of euxinic conditions in basin settings during early T1 times, which became progressively less extreme during T1 deposition. The evolution of redox conditions in basinal settings, and the associated delay in the onset of euxinia at more shallow marginal sites, can be attributed to the interaction of sea-level change with basin paleogeography. Euxinia in the southern Kupferschiefer Sea did not lead to near-quantitative depletion of aqueous Mo, possibly due to short deepwater renewal times in the Thuringian Basin, low aqueous H 2 S concentrations, the continuous resupply of RSTE during transgression and declining burial rates of RSTEs throughout T1 times. Drawdown of RSTE is, however, indicated for euxinic lagoon environments. Moreover, admixture of freshwater supplied to these lagoons by rivers strongly impacted local seawater chemistry. The highest Mo-isotope compositions of ~ 1.70‰ in basin sediments allow a minimum Kupferschiefer Sea seawater composition of ~ 2.40‰ to be estimated. This composition is similar to the ~ 2.30‰ estimate for the Late Permian open ocean, and confirms a strong hydrographic connection between the epeiric Kupferschiefer Sea and the global ocean. The substantial variation in Mo-isotope signatures is paralleled by diagnostic shifts in biomarkers responding to oxygenation in different parts of the water column. Water column chemistry has been affected by variation in sea level, hydrodynamic restriction, riverine freshwater influx and evaporitic conditions in shallow lagoons. Elucidation of the relative role of each driving factor by a single geochemical proxy is not feasible but the complex scenario can be disentangled by a multiproxy approach.

We investigated the proposed necrotic mechanism of ingenol mebutate, a natural compound with anti-cancer properties in human keratinocytes, the human squamous cell carcinoma cell line HSC-5, and HeLa cervix carcinoma cells. Topical application of a clinical dose of ingenol mebutate 0.05% (1.15 mM) gel to human reconstituted full-thickness skin equivalents strongly reduced epidermal, but not dermal viability. Ingenol mebutate showed cytotoxic potency between 200-300 M on normal and cancer cells. When keratinocytes were induced to differentiate, they became significantly less sensitive to ingenol mebutate and half-maximal induction of cell death required more than 300 M ingenol mebutate. Cytotoxic concentrations of ingenol mebutate caused rupture of the mitochondrial network within minutes paralleled by cytosolic calcium release in all cells. Subsequently, plasma membrane integrity was lost as seen by propidium uptake into the cells. This was in sharp contrast to lysis of cells with low concentrations of the detergent Triton X-100 that permeabilized the plasma membrane within minutes without affecting organelle morphology. Buffering of intracellular calcium and inhibition of the mitochondrial permeability transition pore reduced the cytotoxic effect of ingenol mebutate in cancer cells, but not in normal keratinocytes. However, these inhibitors could not prevent cell death subsequent to prolonged incubation. Our findings reveal that ingenol mebutate does not mediate cytotoxicity by a simple lytic, necrotic mechanism, but activates distinct processes involving multiple cell organelles in a cell-type and differentiation-dependent manner. These data improve our understanding of ingenol mebutate-target cell interactions and offer new insights relevant to the removal of aberrant cells in human skin.

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin. ispartof: SCIENTIFIC REPORTS vol:12 issue:1 ispartof: location:England status: published

Actin nucleation is a fundamental biological process involved in several cellular events. Actin filament nucleation requires several hundred nano-molars to micro-molar amounts of polymerizable actin monomers. This requirement makes the impossible use of standard single molecule techniques. Here, we use zero-mode waveguides to reduce the observation volume and monitor, with single molecule resolution, the initial steps in actin filament formation. We immobilized gelsolin, a capping and nucleating protein at the bottom of the waveguides and added up to several micro-molar amounts of fully labeled actin monomers in polymerization conditions. We observe the first steps during filament formation as step-wise increases in detected fluorescence intensity. Mean reaction rates are then estimated for each oligomer size. Arrival time distribution of individual steps reveals the presence of kinetic intermediates. Furthermore, analysis of oligomer size distribution as a function of actin concentration reveals transitions at around 600 and 900 nM where the system dynamics change concomitant with the appearance of filament elongation. The change in dynamics at the transition is associated with an increase in dissociation rates. Such transition is blocked by Latrunculin A, suggesting a functional role for ATP hydrolysis and/or monomer flattening. Our results point towards a gelsolin-mediated actin filament nucleation mechanism where protein-protein interactions trigger ‘nuclei’ instability necessary for filament elongation.