Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of pre‐mRNA splicing. Similar to other classes of nucleic acid‐based therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinyl‐tetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strain‐promoted azide‐alkyne cycloaddition conjugation with splice‐switching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest splice‐switching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2K‐mdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant splice‐switching at very low nanomolar concentrations. The presented aminoethylene‐lipopeptides are thus a promising platform for the generation of PMO‐therapeutics with a favorable activity/toxicity profile.     

Studies of Work ‘in the Wild’

Computer Supported Cooperative Work (CSCW) -     

Ecophysiology and niche differentiation of Nitrospira-like bacteria, the key nitrite oxidizers in wastewater treatment plants.

Nitrite-oxidizing bacteria of the genus Nitrospira are key nitrifiers in wastewater treatment plants. Pure cultures of these organisms are unavailable, but cultivation-independent molecular methods make it possible to detect Nitrospira-like bacteria in environmental samples and to investigate their ecophysiology. Comprehensive screening of natural and engineered habitats and of public databases for 16S rRNA sequences of Nitrospira-like bacteria revealed a surprisingly high biodiversity in the genus Nitrospira, which comprises at least four phylogenetic sublineages. All Nitrospira-like bacteria detected in wastewater treatment plants belonged to the sublineages I and II. Subsequently, the population dynamics of different Nitrospira-like bacteria were monitored, by quantitative fluorescence in situ hybridization with rRNA-targeted probes, confocal laser scanning microscopy and digital image analysis, during incubation of nitrifying activated sludge in media containing different nitrite concentrations. These experiments showed that Nitrospira-like bacteria, which were affiliated with the phylogenetic sublineages I or II of the genus Nitrospira, responded differently to nitrite concentration shifts. Previously unknown properties of Nitrospira-like bacteria were discovered in the course of an environmental genomics project. Implications of the obtained results for fundamental understanding of the microbial ecology of nitrite oxidizers as well as for future improvement of nutrient removal in wastewater treatment plants are discussed.     

Silicon for thin-film transistors

We are standing at the beginning of the industrialization of flexible thin-film transistor (TFT) backplanes. The two important research directions for the TFTs are (i) processability on flexible substrates and (ii) sufficient field-effect mobilities of electrons and holes to support complementary metal insulator semiconductor operation. The most important group of TFT capable semiconductors are the several modifications of silicon films: amorphous, nanocrystalline and microcrystalline. We summarize their TFT properties and their compatibility with foil substrate materials.     

Role of Perceived Importance in Intergroup Contact.

Furthering G. W. Allport's (1954) contentions for optimal contact, the authors introduce a new construct: the perceived importance of contact. They propose that perceived importance is the best proximal predictor of contact's reduction of prejudice. If individuals have opportunities for contact at work or in the neighborhood, their chances to have intergroup acquaintances and friends increase. Intergroup contact among acquaintances and friends can be perceived as more or less important, which in turn determines intergroup evaluations. A 1st study shows that the new measure of perceived importance is indeed distinct from established quantity and quality indicators. The results are cross-validated in a 2nd study that also sheds light on the meaning of importance. In 3rd and 4th studies, structural equation analyses and a meta-analysis support the hypotheses. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of thapsigargin on calcium homeostasis in Trypanosoma cruzi trypomastigotes and epimastigotes

By using the fluorescent calcium indicator fura-2, it was found that the concentration of free Ca2+ in the cytoplasm of Trypanosoma cruzi trypomastigotes incubated in the presence or absence of external calcium was maintained at very low levels (10-20 nM). When trypomastigotes were incubated in the presence of succinate and ATP and permeabilized with digitonin, they lowered the medium calcium concentration to a submicromolar level. In the presence of 1 microM FCCP the initial rate of Ca2+ sequestration by these permeabilized cells was very slow. When succinate alone was present, the initial rate of Ca2+ accumulation was slower than with ATP plus succinate, and the calcium set point was about 0.6 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. High concentrations of the tumor promoter thapsigargin slightly increased cytosolic Ca2+ in the presence of extracellular Ca2+ but had no effect on the FCCP- and oligomycin/antimycin A-insensitive Ca2+ pool. In addition, when used at those concentrations (4-20 microM), thapsigargin was shown to release Ca2+ from the mitochondria and to decrease the inner mitochondrial membrane potential of trypomastigotes and epimastigotes as measured using safranine O. Despite the presence of inositol phosphates as determined by [3H]inositol incorporation, no IP3-sensitive Ca2+ release could be detected in trypomastigotes.