UNICARagil - Disruptive Modular Architectures for Agile, Automated Vehicle Concepts

This paper introduces UNICARagil, a collaborative project carried out by a consortium of seven German universities and six industrial partners, with funding provided by the Federal Ministry of Education and Research of Germany. In the scope of this project, disruptive modular structures for agile, automated vehicle concepts are researched and developed. Four prototype vehicles of different characteristics based on the same modular platform are going to be build up over a period of four years. The four fully automated and driverless vehicles demonstrate disruptive architectures in hardware and software, as well as disruptive concepts in safety, security, verification and validation. This paper outlines the most important research questions underlying the project

Microarrays for the investigation of receptor-ligand-interactions with confocal fluorescence detection

Wechselwirkungen zwischen kleinen Molekülen wurden auf Mikroarrays untersucht. Die eingesetzte Oberflächenchemie ermöglichte gleichzeitig eine einfache Arrayherstellung sowie für spätere Fragestellungen die Unterdrückung des Wachstums adhärenter Zellen außerhalb des Arrays. Die für die Untersuchung gewählte konfokale Fluoreszenzmikroskopie erwies sich als geeignet, sowohl die Oberflächenchemie zu optimieren als auch schwächere Rezeptor-Ligand-Wechselwirkungen zu untersuchen, als sie bei DNA- oder Antikörpermikroarrays üblich sind. So ließ sich die konfokale Detektion in Kombination mit einer Bindungstitration u.a. zur Messung von Bindungskonstanten im Arrayformat für das Modellsystem Vancomycin anwenden. Ebenso ließ sich auf den entwickelten Oberflächen der Aktivierungszustand eines Proteins in Abhängigkeit von einer vorangegangenen Zellstimulation durch Inkubation der Mikroarrays mit unaufgereinigtem Zelllysat untersuchen. Weitere Untersuchungen widmeten sich dem Einfluss unterschiedlicher Fluorophore auf die beobachteten Wechselwirkungen sowie dem Einfluss der Rezeptordichte auf die Bindungsstärke.Interactions between small molecules were investigated on microarrays. The applied surface chemistry allowed a simple generation of the arrays and at the same time, for future questions, the suppression of cell growth outside the arrays. Confocal fluorescence microscopy proved to be useful for both optimisation of the surface chemistry and investigation of receptor-ligand-interactions weaker than those usually occurring on DNA or antibody microarrays. In combination with binding titrations, confocal detection could be applied to determine binding constants in an array format for vancomycin as model system. Also, the activation state of a protein, depending on a preceding stimulation of cells, could be investigated by incubating microarrays with crude cell lysate. Further experiments determined the influence of different fluorophores on the observed interactions and the influence of receptor density on the strength of binding

Peptide microarrays for the detection of molecular interactions in cellular signal transduction.

The formation of protein complexes is a hallmark of cellular signal transduction. Here, we show that peptide microarrays provide a robust and quantitative means to detect signalling-dependent changes of molecular interactions. Recruitment of a protein into a complex upon stimulation of a cell leads to the masking of an otherwise exposed binding site. In cell lysates this masking can be detected by reduced binding to a microarray carrying a peptide that corresponds to the binding motif of the respective interaction domain. The method is exemplified for the lymphocyte-specific tyrosine kinase 70 kDa zeta-associated protein binding to a bis-phosphotyrosine-motif of the activated T-cell receptor via its tandem SH2 domain. Compared to established techniques, the method provides a significant shortcut to the detection of molecular interactions

Multivalence and spot heterogeneity in microarray-based measurement of binding constants.

Item does not contain fulltextMicroarray technology is increasingly used for a miniaturised and parallel measurement of binding constants. In microarray experiments heterogeneous functionalization of surfaces with capture molecules is a problem commonly encountered. For multivalent ligands, especially, however, binding is strongly affected by receptor density. Here we show that high-resolution imaging of microarrays followed by image segmentation and separate analysis of bright and dark parts provides valuable information about ligand binding. Binding titrations were conducted with monovalent and bivalent fluorescent ligand peptides for the model receptor vancomycin. Microarrays were scanned with a confocal microscope and inhomogeneous spots were evaluated either as a whole or after segmentation into bright and dark areas. Whereas the binding constant for the monovalent ligand was hardly affected by spot heterogeneity, for the bivalent ligand affinity was higher for the parts of the spots with a greater density of receptors. This information was lost if the spots were analysed as a whole. These results reveal that imaging resolution may be a key factor in miniaturised binding assays, emphasising the importance of high-resolution images and image segmentation for new techniques, for example SPR imaging

Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers

Synthetic nanoparticle formulations have the potential for tumor-targeted gene delivery. Receptor-targeted nanocomplex (RTN) formulations comprise mixtures of cationic liposomes and targeting peptides that self-assemble on mixing with nucleic acids. RTN formulations were prepared containing different polyethylene glycol (PEG)ylated lipids with esterase-cleavable linkers (e. g., ME42) to promote intracellular PEG detachment and nanoparticle disassembly. In addition, integrin-targeting peptides (peptide ME27) were tested with endosomal furin-and cathepsin B-cleavable peptide linkers located between the integrin-binding ligand and the K-16 nucleic acid-binding domain to promote intracellular disengagement from the receptor. ME42/ME27 RTNs formed stable particles of <200 nm in isotonic salt buffers, compared with 4-mu m particles formed by un-PEGylated RTNs. Transfection efficiency by PEG-modified, cleavable RTNs improved similar to 2-fold in 4 different cell lines, with 80% efficiency in murine neuroblastoma cells. In an in vivo model of neuroblastoma, ME42/ME27 RTNs delivering luciferase genes were tumor specific, with little expression in other organs tested. PEGylation of the RTNs enhanced luciferase transfection 5-fold over non-PEG formulations, whereas the cleavability of the peptide ME27 enhanced transfection 4-fold over that of RTNs with noncleavable peptides. Cleavability of the lipid for in vivo transfections had no effect. PEGylated, cleavable RTN formulations offer prospects for tumor-specific therapeutic gene transfer.-Grosse, S. M., Tagalakis, A. D., Firouz Mohd Mustapa, M., Elbs, M., Meng, Q.-H., Mohammadi, A., Tabor, A. B., Hailes, H. C., Hart, S. L. Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers. FASEB J. 24, 2301-2313 (2010). www.fasebj.or

A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG ( E), and the cationic liposomes ( L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid ( D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED- 2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF

Stabilised integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM