Beurteilung der geomechanischen Stabilität und Integrität von Endlagerbergwerken im Salzgebirge auf der Grundlage geologischer und ingenieurgeologischer Untersuchungen

Any safety assessment of a permanent repository for radioactive waste has to include an analysis of the geomechanical stability of the repository and integrity of the geological barrier. Such an analysis is based on geological and engineering geological studies of the site, on laboratory and in-situ experiments, and on numerical calculations. Central part of the safety analysis is the geomechanical modelling of the host rock. The model should simulate as closely as possible the conditions at the site and the behaviour of the rock (e.g., geology, repository geometry, initial rock stress, and constitutive models). On the basis of the geomechanical model numerical calculations are carried out using the finite-element method and an appropriate discretization of the repository and the host rock. The assessment of the repository stability and the barrier integrity is based on calculated stress and deformation and on the behaviour of the host rock measured and observed in situ. An example of the geomechanical analysis of the stability and integrity of the Bartensieben mine, a former salt mine, is presented. This mine is actually used as a repository for low level radioactive waste. The example includes all necessary steps of geological, engineering geological, and geotechnical investigations.Die erforderlichen Sicherheitsnachweise für Endlager für radioaktive Abfälle umfassen insbesondere die geomechanische Beurteilung der Standsicherheit der Anlage und der Integrität der geologischen Barriere. Dazu sind geologische und ingenieurgeologische Standortuntersuchungen, Laborversuche, In-situ-Untersuchungen und numerische Berechnungen erforderlich. Kernstück des Sicherheitsnachweises ist die Erstellung eines geomechanischen Modells, das möglichst realitätsnah die Situation am Endlagerstandort und das Gebirgsverhalten (z.B. geologische Struktur, Hohlraumgeometrie, primäre Gebirgsspannungen, Materialmodelle) beschreibt. Auf der Grundlage des geomechanischen Modells ist ein Finite-Elemente-Modell zu erstellen, das eine zweckmäßige Diskretisierung des betrachteten Gebirgsbereiches und die Wahl geeigneter Randbedingungen und Lasteinwirkungen umfasst. Mit diesem Modell werden die durch das Endlager im Gebirge verursachten Spannungs- und Verformungszustände berechnet und zusammen mit den Ergebnissen aus In-situ-Messungen, aus örtlichen Beobachtungen und aus Laborversuchen zur Beurteilung der Stabilität und Integrität des Endlagers herangezogen. Die prinzipielle Methodik eines Integritätsnachweises wird exemplarisch für das ehemalige Salzbergwerk Bartensieben erläutert, das als Endlager für schwach radioaktive Abfälle genutzt wird. Dazu werden die erforderlichen geologischen Grundlagen und das daraus abgeleitete geomechanische Modell sowie die in Modellberechnungen ermittelten Spannungs- und Verformungszustände im Gebirge dargestellt. Stabilität und Integrität der Salzbarriere werden anhand dieser Ergebnisse und verschiedener geomechanischer Kriterien bewertet

Characterization of cell uptake and intracellular trafficking of exosomes by quantitative live cell imaging: towards biomimetic delivery vehicles of therapeutic RNA

Exosomes are biological nanoparticles which play a role in long distance cell-to cell communication. These 40-100 nm sized vesicles are released by virtually all cells and derive from the multivesicular bodies within their parent cells. They modulate their target cell fate by induction of cell signaling as well as RNA and protein cargo transfer. Exosomes have also moved into the spotlight of clinical research, with potential use as biomarkers or next generation therapeutic delivery agents. Exosomes are thought to be highly efficient intercellular messengers but quantitative characterization is lacking. Also, their routes of cell uptake and subcellular fate within recipient cells remain elusive. This work introduces an in depth and quantitative characterization of exosome cargo, physicochemical properties, labeling, isolation and their recipient cell interaction at the single cell – single vesicle level. Basic protocols for exosome purification were revisited in order to allow for isolation of exosomes with sufficient yields and in as native state as possible to enable functional studies. Since exosome integrity and recovery yields after differential ultracentrifugation (UC), the most commonly used protocol for exosome isolation, turned out to be poor and unreproducible, we describe an alternative protocol based on ultrafiltration (UF) with subsequent gel filtration (GF) for recovering exosomes relatively selectively, with intact biophysical and functional properties and significantly higher yields. Next we establish methods for specific exosome labeling using fluorescent marker proteins transiently expressed in parent cells, which led to a focus on FP tagged CD63 constructs. CD63-emGFP labeled exosomes were extensively characterized and showed identical properties compared to unlabeled exosomes based on sucrose density gradient, CryoTEM microscopy and proteomics analysis. Furthermore, we successfully adapted fluctuation correlation spectroscopy for characterization of fluorescently labeled exosomes. In another part of this work we describe a high content screen for exosome uptake which we use to provide a first systematic and quantitative profiling of exosome uptake across a panel of exosome parent recipient cells, including HEK293, Huh7, B16F10 as parental cells and additional primary fibroblasts, primary keratinocytes, iPS derived motor neurons and HUVEC primary human endothelial cells as recipient cell lines. These quantitative profiling data reveals preferences in exosome internalization by different cell types and suggests that specific receptor ligand interactions may determine tissue specificity. Finally, we address one of the fundamental questions in the field of cellular communication: how exosomes released by one cell enter and interact with their recipient cell. Our data quantifies for the first time the cell uptake dynamics of exosomes at the single vesicle and single cell level and reveals a quantitative efficiency paralleling that of infective pathogens rather than artificial delivery vehicles. We demonstrate that exosome uptake is largely mediated by active recruitment and surfing on filopodia to reach endocytic hotspots for their internalization at the filopodia base. This provides a cell biological explanation for the remarkably high efficiency of exosomes in targeting recipient cells and discovers a new parallel to some viruses and other pathogens. We propose that the process of filopodia surfing may have evolved as a highway for exosomes into the cell, being hijacked by certain pathogens for host cell interaction. This data does not support the previously reported exosome uptake by vesicle fusion with the plasma membrane or cargo release by endosomal escape. Instead we observe intact exosome uptake to enter endocytic vesicles, which then scan along the endoplasmic reticulum (ER) and end up in lysosomes. Our data suggest a model of controlled cargo delivery to defined subcellular localizations like the ER, rather than vesicle fusion and free release into the cytoplasm

Development of the chick wing and leg neuromuscular systems and their plasticity in response to changes in digit numbers

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations

Perinatal health care of women without health care insurance in Germany

In Germany, approximately 60,000 people live without health insurance, including women of reproductive age. Pregnant and childbearing women and their children in this group are exposed to greater health risks. To ensure their care, midwives and gynecologists offer voluntary and free services as part of interdisciplinary teams. Even if regional solutions exist, national regulations have to be developed and implemented

Adipose mTORC2 is essential for sensory innervation in white adipose tissue and whole-body energy homeostasis

Adipose tissue, via sympathetic and possibly sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue are poorly characterized.; Taking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue.; Our findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results also suggest that WAT may affect whole-body energy homeostasis via sensory neurons

Adipose mTORC2 is essential for arborization of sensory neurons in white adipose tissue and whole-body energy homeostasis

Adipose tissue, via sympathetic and sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue is poorly characterized. Taking advantage of recent progress in whole-mount three-dimensional imaging of adipose tissue, we identified a neuronal network of calcitonin gene-related protein (CGRP)-positive sensory neurons in white adipose tissue (WAT). Furthermore, we show that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, mediates sensory innervation in WAT. Based on visualization of neuronal networks, mTORC2-deficient WAT displayed reduced arborization of (CGRP)-positive sensory neurons, while sympathetic neurons were unaffected. This selective loss of sensory innervation followed reduced expression of growth-associated protein 43 (GAP43) in CGRP-positive sensory neurons. Finally, we found that loss of sensory innervation in WAT correlated with systemic insulin resistance. Our findings suggest that adipose mTORC2 is necessary for sensory innervation in WAT which likely contributes to WAT-to-CNS communication

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation

Lymphotoxin α/β and Tumor Necrosis Factor Are Required for Stromal Cell Expression of Homing Chemokines in B and T Cell Areas of the Spleen

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) α/β lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTα- and LTβ-deficient mice. Treatment of adult mice with antagonists of LTα1β2 also leads to decreased BLC expression. These findings indicate that LTα1β2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTα-, and LTβ-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus–induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTα1β2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Extracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV-related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV-producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40–60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluorescent EVs to link single vesicle analysis with single molecule quantification

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications