Effective zero-thickness model for a conductive membrane driven by an electric field

The behavior of a conductive membrane in a static (DC) electric field is investigated theoretically. An effective zero-thickness model is constructed based on a Robin-type boundary condition for the electric potential at the membrane, originally developed for electrochemical systems. Within such a framework, corrections to the elastic moduli of the membrane are obtained, which arise from charge accumulation in the Debye layers due to capacitive effects and electric currents through the membrane and can lead to an undulation instability of the membrane. The fluid flow surrounding the membrane is also calculated, which clarifies issues regarding these flows sharing many similarities with flows produced by induced charge electro-osmosis (ICEO). Non-equilibrium steady states of the membrane and of the fluid can be effectively described by this method. It is both simpler, due to the zero thickness approximation which is widely used in the literature on fluid membranes, and more general than previous approaches. The predictions of this model are compared to recent experiments on supported membranes in an electric field.Comment: 14 pages, 5 figure

Dendritic Core-Multishell Nanocarriers in Murine Models of Healthy and Atopic Skin

Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e.g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment. Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection. Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry- assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis. Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin

DYAMOND: the DYnamics of the Atmospheric general circulation Modeled On Non-hydrostatic Domains

A review of the experimental protocol and motivation for DYAMOND, the first intercomparison project of global storm-resolving models, is presented. Nine models submitted simulation output for a 40-day (1 August–10 September 2016) intercomparison period. Eight of these employed a tiling of the sphere that was uniformly less than 5 km. By resolving the transient dynamics of convective storms in the tropics, global storm-resolving models remove the need to parameterize tropical deep convection, providing a fundamentally more sound representation of the climate system and a more natural link to commensurately high-resolution data from satellite-borne sensors. The models and some basic characteristics of their output are described in more detail, as is the availability and planned use of this output for future scientific study. Tropically and zonally averaged energy budgets, precipitable water distributions, and precipitation from the model ensemble are evaluated, as is their representation of tropical cyclones and the predictability of column water vapor, the latter being important for tropical weather

Tropical cyclones in global storm-resolving models

Recent progress in computing and model development has initiated the era of global storm-resolving modeling and with it the potential to transform weather and climate prediction. Within the general theme of vetting this new class of models, the present study evaluates nine global-storm resolving models in their ability to simulate tropical cyclones (TCs). Results show that, broadly speaking, the models produce realistic TCs and remove longstanding issues known from global models such as the deficiency to accurately simulate TC intensity. However, TCs are strongly affected by model formulation, and all models suffer from unique biases regarding the number of TCs, intensity, size, and structure. Some models simulated TCs better than others, but no single model was superior in every way. The overall results indicate that global storm-resolving models are able to open a new chapter in TC prediction, but they need to be improved to unleash their full potential

Anwendung und Zelluläre Interaktionen von Kationischen Nanopartikeln

1 Introduction 5 1.1 Nanoparticles 5 1.2 Advantages and Applications of Cationic Nanoparticles 8 1.2.1 Enhanced Cellular Uptake and Endosomal Escape 8 1.2.2 Complexation of Nucleic Acids 11 1.3 Adverse Effects of Nanoparticles 13 1.3.1 Systemic Effects 13 1.3.2 Cellular and Subcellular Mechanisms 15 1.4 Strategies to overcome adverse effects 22 1.4.1 Nanoparticle Materials 22 1.4.2 Biodegradability 24 2 Scientific Goal 26 3 Publications and Manuscripts 28 3.1 Systematic Adjustment of Charge Densities and Size of Polyglycerol Amines Reduces Cytotoxic Effects and Enhances Cellular Uptake 28 3.2 Crosslinked Redox-Responsive Micelles Based on Lipoic Acid-Derived Amphiphiles for Enhanced siRNA Delivery 42 3.3 Synthesis of pH-Cleavable dPG-Amines for Gene Delivery Application 71 3.4 Defined pH-Sensitive Nanogels as Gene Delivery Platform for siRNA Mediated in vitro Gene Silencing 106 3.5 FLIM-ROX as a Highly Sensitive Fluorescence Lifetime Based Approach for Reliable Reactive Oxygen Species Detection in vitro and in vivo 122 4 Conclusions and Outlook 163 5 Kurzzusammenfassung 165 6 References 168 7 Appendices 179 7.1 List of other Publications 179 7.2 Abbreviations 180The goals of this work were the development of new cationic carrier systems that are able to transport sensitive nucleic acid cargo into cells while at the same time de-creasing their adverse effects. This was realized with the use of PG as the polymeric backbone of the carrier systems and a cleavable linker that enables the particles bio-degradability in the cytosol and also a release of the cargo material. The first project was a systematic approach to find a best candidate in the frame of hyperbranched polyglycerol nanocarriers by tweaking both the size and the hydroxyl-to-amino ratio on the particle surface. The particles were tested for their performance to transfect plasmid DNA in vitro. Among several candidates, PG with a size of 14 kDa and 90 % of amine surface groups and PG 200 kDa with 30 % amine groups were the most promising candidates. These findings are in accord with those of Zeng et al., where 14 kDa particles worked best with 50 % amines and 55 kDa particles worked best with 35 % amines for siRNA delivery. The best candidate with similar transfection efficacies as PEI, 200 kDa HPG and 30 % amines, were further studied to elaborate their exact mechanism of toxicity. Compared to 25 kDa branched PEI (bPEI), the cell viability was less affected. Additional tests revealed no evidence for apoptosis but a slight membrane disruption which was more prominent in PEI than in the HPG. In the second project, micelles based on lipoic acid and PG were combined with a redox sensitive disulfide crosslinker for the delivery of siRNA. All biocompatible build-ing blocks were selected so that there will be no toxicity upon particle degradation and four different structure were generated. Acceptable cell viabilities (> 70 %) were ob-served for all but one construct up to an N/P ratio of 120. One of the three candidates showed comparable transfection efficacy to the commercial control Lipofectamine® but was still lower. Two other approaches featured the efficacy of different PG poly-mer structures for transfection: hyperbranched PG with an amine shell and a PG nano-gel with small 600 Da PEI units. Both these systems incorporated a pH sensitive cleav- able linker. The HPG carriers contained a cleavable linker (50 % cleaved after 12 hours) and a fast-cleavable variety of that (50 % cleaved after 4 hours). The cell viabil-ity of the non-degradable carrier and the two degradable ones was comparable and the cleavable one also showed similar transfection efficacy. However, the fast-cleavable carriers showed a decreased performance, indicating that the degradation process needs to be delayed for efficient transfection. The other benzacetal-linker sys-tem was a crosslinked nanogel comprising linear PG and small linear PEI molecules. This system was able to physically encapsulate siRNA during the synthesis process and showed a transfection efficacy similar to that of 25 kDa bPEI. Also, the cell viability was higher, especially at higher concentrations and a subsequent test using red blood cells showed high biocompatibility with biological membranes, whereas PEI showed a visi-ble disruptive effect. The nanogel approach also eliminated the need for complexation of nucleic acids and the carrier system prior to application, making them more feasi-ble in practical use. This feature and the increased biocompatibility makes these sys-tem promising candidates for further in vivo tests. Another project focused on the sensitive detection of reactive oxygen species upon nanoparticle exposure. For this, fluorescence lifetime imaging microscopy (FLIM) was utilized because it increased the sensitivity of the commercial CellROX Green® dye. Calibration curves proved the high functionality of the method in fixed and live cells. Further studies on ex vivo skin tissue demonstrated the methods applicability for more complex systems. Also, to consolidate the picture that the ROS generation by model particles made of bioinert gold with an amine shell, other biological studies were per-formed. These studies revealed induction of cellular senescence and genotoxic effect at subtoxic concentrations as determined by cell viability studies. The results confirm the overall picture of cationic nanoparticle induced adverse effects on cells and legiti-mate this FLIM method for further routine application for toxicological tests. For the future, these polycationic nanoparticle delivery system will be further ex- plored for their in vitro and in vivo application of nucleic acid delivery, or gene deliv-ery, and the delivery of other drugs. The challenge of outperforming viral carrier sys-tems or decreasing the adverse effects completely have not yet been entirely accom-plished. Further work will be done on the nanogels due to their flexible size range and the ability to encapsulate a great variety of different cargoes. Also, the interactions of nanoparticles and cells regarding adverse effects needs to be further elucidated. As a sensitive method for the detection of ROS was established in the last project using FLIM, further effort can be taken to eliminate subtoxic oxidative stress and possibly outperform current drug delivery systems.Das Ziel dieser Arbeit bestand aus der Entwicklung kationischer Nanopartikel- Trägersysteme, die in der Lage sind, sensible Gast-Moleküle wie Nukleinsäuren in Zel-len zu transportieren und gleichzeitig ihre adversen Effekte zu reduzieren. Realisiert wurde das durch die Verwendung von Polyglycerin als Polymergrundlage des Träger-systems und spaltbarer, Stimuli-responsiver Querverbindungen. Diese ermöglichten die Bioabbaubarkeit der Partikel im Zytosol und den Lysosomen und die Freisetzung der Gastmoleküle. Das erste Projekt bestand aus einer systematischen Herangehensweise, um das beste Konstrukt eines hochverzweigten, Polyglycerin-basierten Nanoträgers zu finden. Hierzu wurden sowohl die Größe als auch das Verhältnis von Hydroxyl- zu Aminogrup-pen auf der Partikeloberfläche moduliert. Anschließen wurden die Partikel auf ihr Fä-higkeit getestet, in vitro Plasmid DNA in Zellen zu transfizieren. Unter mehreren Kan-didaten stellten sich PG mit einer Größe von 14 kDa mit 90 % Aminogruppen und 200 kDa mit 30 % Aminogruppen als die besten heraus. Damit konnten die Ergebnisse von Zeng et al bestätigt werden, wonach 14 kDa Partikel mit 50 % Aminogruppen und 55 kDa partikel mit 35 % Aminogruppen am besten für die Transfektion von siRNA geeig-net waren.[152] Der beste Kandidat mit einer vergleichbaren Transfektionseffizienz wie PEI, 200 kDa HPG mit 30 % Aminogruppen, wurde des Weiteren auf die Mechanismen der Toxizität geprüft. Im Vergleich zu 25 kDa bPEI (bPEI) war die Zellviabilität allge-mein weniger beeinträchtigt. Zusätzliche Tests zeigten keine Anzeichen von Apoptose-Induktion, aber eine leichte Störung der Membranintegrität, wobei der Effekt durch PEI deutlicher ausgeprägt war. Im zweiten Projekt wurden Liponsäure und PG mit einer redox-sensitiven Verknüp-fung kombiniert, um siRNA zu transfizieren. Alle Grundbausteine wurden so gewählt, dass bei einer Spaltung keine toxischen Abbauprodukte freigesetzt werden und vier verschiedene Konstrukte untersucht. Für drei von vier Konstrukte wurde eine akzeptab-le Zellviabilität festgestellt, bis zu einem N/P-Verhältnis von 120. Einer der drei Kandi-daten zeigte eine Transfektioneffizienz, die mit der kommerziell erhältlichen Kontrolle Lipofectamine ® vergleichbar war, wenn auch etwas niedriger. Zwei andere Herange-hensweisen befassten sich mit der Effizienz anderer PG Polymerkonstrukte für Trans-fektionen: hochverzweigtes PG mit einer Amin-Schale und ein PG-Nanogel mit kleinen 600 Da PEI Einheiten. Beide Systeme beinhalten eine pH-sensitiven Querverbindung. Die HPG Träger wurden mit einer spaltbaren Queverbindung und einer schnell-spaltbaren Variante davon ausgestattet, wobei 50 % der Träger nach 12 Stunden bzw. 4 Stunden gespalten wurden. Die Zellviabilität des Trägers ohne spaltbare Querverbin-dung und dieser beiden Konstrukte waren sehr ähnlich, aber nur das spaltbare zeigte eine vergleichbare Transfektionseffizienz zu dem nicht- spaltbaren. Die schnell-spaltbare Variante allerdings zeigte eine schlechtere Performance, was darauf hindeu-tet, dass der Spaltungsvorgang für eine effiziente Transfektion langsamer von statten gehen muss. Das andere pH- sensitive, Benzazetal-verknüpfte System bestand aus Na-nogelen bestehend aus PG und kleinen, linearen PEI Einheiten. In diesem System konn-te siRNA während der Synthese physikalisch eingekapselt werden. Die Transfektioneffi-zienz dieses Konstrukts war vergleichbar, wenn auch niedriger, mit der von 25 kDa bPEI. Andererseits war die Zellviabilität höher, vor allem bei hohen Konzentrationen. Ein zusätzlicher Test an roten Blutzellen demonstrierte eine hohe Biokompatibilität gegenüber biologischen Membranen, während PEI einen deutlichen schädlichen Effekt zeigte. Der Nanogel-Ansatz als Träger-System eliminiert außerdem die Notwendigkeit für eine Komplexierung von Nukleinsäuren vor der Anwendung, da diese bereits ver-kapselt sind. Das macht sie in der Anwendung vergleichsweise praktikabel. Insgesamt machen die Vorteile der erhöhten Biokompatibilität und der Einfachheit der Anwen-dung dieses System zu einem vielversprechenden Kandidaten für spätere in vivo Versu-che. Ein anderes Projekt war auf die sensitive Detektion von reaktiven Sauerstoffspe- ziesfokussiert, welche bei Kontakt mit Nanopartikeln freigesetzt werden können. Dafür wurde der Farbstoff CellROX Green ® in Kombination mit Fluorescence Lifetime Ima-ging Microscopy (FLIM) verwendet, um dessen Sensitivität zu erhöhen. Durch Kalibra-tionskurven konnte die Funktionalität der Methode sowohl in fixierten als auch in le-benden Zellen gezeigt werden. Des Weiteren konnte die Anwendbarkeit der Methode für komplexere biologische Systeme anhand von ex vivo Versuchen demonstriert wer-den. Um das Gesamtbild um den gemessen oxidativen Stress zu vervollständigen, wur-den Modell-Partikel aus Gold mit einer Amin-Schale verwendet und weitere biologi-sche Untersuchungen durchgeführt. Dazu wurden Zellviabilitätsstudien durchgeführt und subtoxische Konzentrationen verwendet. Diese demonstrierten neben den der Ge-nerierung von ROS auch die Induktion von zellulärer Seneszenz und einen genotoxi-schen Effekt. Die Ergebnisse bestätigten das allgemeine Gesamtbild der adversen Ef-fekte von kationischen Nanopartikeln auf Zellen und legitimierten damit diese neue FLIM-Methode und deren möglichen Einsatz in Routine-Kontrollen im Zusammenhang mit medizinischen Anwendungen. Für die Zukunft werden diese vielversprechenden kationischen Nanopartikel- Trägersysteme weiter erforscht, sowohl für die Anwendung mit Nukleinsäuren als auch anderen Arzneistoffen und das sowohl in vitro als auch in vivo. Die Herausforderung, virale Vektorsysteme zu übertreffen oder die adversen Effekte komplett zu eliminie-ren, ist noch nicht gemeistert. Weitere Arbeit sollte in die Nanogel-Systeme gesteckt werden, da diese durch ihre flexible Größe und der Möglichkeit zur Einkapselung ver-schiedenster Arzneimittel ein besonders großen Potential haben. Zusätzlich sollten die Interaktionen zwischen kationischen Nanopartikeln und Zellen sowie die adversen Ef-fekte weiter aufgeklärt werden. Da mit dieser Arbeit eine sensitive Methode für die Messung von ROS mittels FLIM-Technologie etabliert wurde, sollte diese genutzt wer- den, um die adversen Effekte bei toxischen und subtoxischen Konzentrationen weiter zu eliminieren. Auf lange Sicht könnte das eine aussagekräftige Methode sein, aktuelle Trägersysteme weiter zu untersuchen und zu verbessern

Tissue specificity of the initiation of immunoglobulin k gene transcription

Falkner FG, Neumann E, Zachau HG. Tissue specificity of the initiation of immunoglobulin k gene transcription. Hoppe-Seyler's Zeitschrift für physiologische Chemie. 1984;365:1331-1343.The transient transcription of a rearranged mouse immunoglobulin k gene was studied in a monkey fibroblast cell line. The gene was inserted into an SV40 expression vector and the calcium phosphate coprecipitation method was used for transfection. The transcripts were correctly spliced; transcription, however, was initiated within the vector and not at the correct site 23-26 bp upstream of the gene, irrespective of the length of the upstream sequences (90, 160, 370, and 870 bp) in the plasmid constructs. In contrast, accurately initiated transcripts were observed when a plasmid containing the k gene with 870 bp of its upstream sequence was introduced into a lymphoid cell line; the plasmid was constructed from the pSV2-gpt vector and the electric impulse method was used fro gene transfer in most experiments. Tissue-specific expression of k light chain genes in lymphoid cells is known to depend on the presence of an enhancer element in the J-C intron. The results reported in this paper suggest that the sequence elements pd and dc which are located upstream of the leader gene segment also act in a tissue-specific manner ant that it is the initiation of transcription which is a tissue-specific event

Potential Analysis Of Flexible Small Series Production Of Spare Parts By Direct Polymer Additive Tooling

In recent decades, there has been an increasing creation of vehicle model variants and product individualization for customer needs. There is a need for a greater variety of spare parts which is leading to high requirements on their logistics. As a result of this trend, automotive production companies have to ensure the provision of many different spare parts as part of their post-fulfillment obligations. However, the surplus spare parts stock will increase the operation cost of the company. This implies the need for a more flexible approach to spare parts production and provision to improve profitability compared to existing process chains. In this research paper, the potential of flexible spare parts production and provision is discussed. For this purpose, the state of spare part production technologies and existing approaches for spare part service are analyzed regarding their technological characteristics, flexibility as well as cost structure. Following this, an approach for flexible production for spare parts with regard to its potential is analyzed and demonstrated based on three exemplary automotive use cases using additively manufactured production resources. In the casting use case, a Fused Filament Fabricated (FFF) additively manufactured speedometer screw model will be used as a sand casting form. The production of deep-drawn car body parts using polymer-based FFF forming tools is investigated in the second use case. Lastly, the production of ignition distributor caps by PolyJet Modelling (PJM) manufactured Injection Molding (PUR-RIM) molds is presented