A general approach applicable to other radiation sources and biological targets

The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine geant4 particle- scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E1/2=6±4 eV. The presented method is applicable for ionizing radiation (e.g., γ rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells

Grundlagen, Strategien und Techniken der Anonymisierung von Transkripten in der qualitativen Forschung: eine praxisorientierte Einführung

In der qualitativen Forschungspraxis müssen beim Datenschutz die gesetzlichen Rahmensetzungen beachtet werden; damit einhergehende Fragen der Anonymisierung gehen für qualitativ Forschende jedoch darüber hinaus. Denn will man Beforschte vor negativen Folgen durch eine Teilnahme schützen und dabei zugleich die eigenen Erkenntnismöglichkeiten ausschöpfen, müssen fall- und kontextspezifische Textveränderungen und andere Maßnahmen in den verschiedenen Phasen empirischer Praxis abgewogen werden. Dabei stellt das konkrete Vorgehen des Anonymisierens selbst jedoch häufig eine Blackbox dar, die in wissenschaftlichen Veröffentlichungen eher rudimentär Beachtung findet. Mit diesem Artikel stellen wir daher nicht nur die aktuellen rechtlichen Rahmenbedingungen dar, sondern fokussieren insbesondere die praktischen Aspekte des Anonymisierens von Transkripten. Mit dem Ziel, einen grundlegenden, zugänglichen und praktisch orientierten Einstieg in das Thema zu geben, systematisieren wir zunächst Datenarten, Anonymisierungsstrategien, und -techniken. Anschließend diskutieren wir praktische Umsetzungsmöglichkeiten und geben Interessierten die Möglichkeit, ein fiktives Transkript selbstständig zu bearbeiten bzw. in Workshops zum Thema einzubinden. Abschließend präsentieren wir grundlegende Schlussfolgerungen für die Methodenausbildung und plädieren für einen offenen kollegialen Austausch zu Anonymisierungspraktiken.In the practice of qualitative research, legal frameworks must be considered in terms of data protection. These frameworks have implications for qualitative researchers regarding the anonymisation of qualitative data. In order to protect research participants from possible negative ramifications of their involvement, yet maximize the insights, case and context specific alterations to transcripts and other measures must be weighed during different stages of research. However, the precise practices of anonymization often remain a black box, due to few discussions in scientific publications. In this article, we provide recent legal frameworks, and also focus on these practices of anonymization. In order to present a foundational, accessible, and practice-oriented introduction, we differentiate various kinds of data, strategies of anonymization, and concrete techniques. Then, we discuss practical implementations and provide a fictive transcript for independently rehearsing these techniques or utilizing it in workshops. We end by formulating conclusions for educating future researchers, and plead for an open collegial exchange on practices of anonymization

The Evolution of Complex Muscle Cell In Vitro Models to Study Pathomechanisms and Drug Development of Neuromuscular Disease

Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover relevant disease mechanisms and enhance the translation of therapeutic findings to strengthen neuromuscular disease precision medicine. By concentrating on idiopathic inflammatory muscle disorders, we summarize the recent evolution of the novel in vitro models to study disease mechanisms and therapeutic strategies. A particular focus is laid on the integration and simulation of multicellular interactions of muscle tissue in disease phenotypes in vitro. Finally, the requirements of a neuromuscular disease drug development workflow are discussed with a particular emphasis on cell sources, co-culture systems (including organoids), functionality, and throughput.Peer Reviewe

Molecular Mechanisms

Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free- vibrational density of states of water due to ectoine, as well as the use of ectoine as an hydroxyl-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR)

Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods

Regioselective functionalization of tetrabromophenanthroline-ruthenium complexes

Structural, photophysical and -chemical characterisation and reactivity of a novel polypyridyl ruthenium complex based on 3,5,6,8-tetra-bromophenanthroline are discussed. Signal storage at a molecular level is great challenge for chemistry.1 The possibility of connecting different functionalities selectively to one ligand of a metal complex may open the route towards higher integrated molecular units capable of processing various external stimuli in a predesignated order. The implementation of this concept demands ligands with a multitude of potential connecting groups which can selectively be transformed.2 3-bromo- and 3,8-dibromophenanthrolines have proved useful for the preparation of mononuclear3 and multiheteronuclear complexes.4 These systems have found applications ranging from DNA photoprobes5 to metalloligands in catalysis.6 A very useful feature of this bromophenanthroline ruthenium complexes is their susceptibility towards nucleophilic aromatic substitution which is very well established

Measurements and simulations of microscopic damage to DNA in water by 30 keV electrons: A general approach applicable to other radiation sources and biological targets

The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine geant4 particle-scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E1/2=6±4 eV. The presented method is applicable for ionizing radiation (e.g., γ rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells

Implications for the Binding of the Protein G5P to DNA

Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration- dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms

The chemokine receptor CXCR5 is pivotal for ectopic mucosa-associated lymphoid tissue neogenesis in chronic Helicobacter pylori-induced inflammation

Ectopic lymphoid follicles are a key feature of chronic inflammatory autoimmune and infectious diseases, such as rheumatoid arthritis, Sjögren's syndrome, and Helicobacter pylori-induced gastritis. Homeostatic chemokines are considered to be involved in the formation of such tertiary lymphoid tissue. High expression of CXCL13 and its receptor, CXCR5, has been associated with the formation of ectopic lymphoid follicles in chronic infectious diseases. Here, we defined the role of CXCR5 in the development of mucosal tertiary lymphoid tissue and gastric inflammation in a mouse model of chronic H. pylori infection. CXCR5-deficient mice failed to develop organized gastric lymphoid follicles despite similar bacterial colonization density as infected wild-type mice. CXCR5 deficiency altered Th17 responses but not Th1-type cellular immune responses to H. pylori infection. Furthermore, CXCR5-deficient mice exhibited lower H. pylori-specific serum IgG and IgA levels and an overall decrease in chronic gastric immune responses. In conclusion, the development of mucosal tertiary ectopic follicles during chronic H. pylori infection is strongly dependent on the CXCL13/CXCR5 signaling axis, and lack of de novo lymphoid tissue formation attenuates chronic immune responses

Lens stem cells may reside outside the lens capsule: an hypothesis

In this paper, we consider the ocular lens in the context of contemporary developments in biological ideas. We attempt to reconcile lens biology with stem cell concepts and a dearth of lens tumors