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.     

Patterning of the chick forebrain anlage by the prechordal plate

We analysed the role of the prechordal plate in forebrain development of chick embryos in vivo. After transplantation to uncommitted ectoderm a prechordal plate induces an ectopic, dorsoventrally patterned, forebrain-like vesicle. Grafting laterally under the anterior neural plate causes ventralization of the lateral side of the forebrain, as indicated by a second expression domain of the homeobox gene NKX2.1. Such a lateral ventralization cannot be induced by the secreted factor Sonic Hedgehog alone, as this is only able to distort the ventral forebrain medially. Removal of the prechordal plate does not reduce the rostrocaudal extent of the anterior neural tube, but leads to significant narrowing and cyclopia. Excision of the head process results in the caudal expansion of the NKX2.1 expression in the ventral part of the anterior neural tube, while PAX6 expression in the dorsal part remains unchanged. We suggest that there are three essential steps in early forebrain patterning, which culminate in the ventralization of the forebrain. First, anterior neuralization occurs at the primitive streak stage, when BMP-4-antagonizing factors emanate from the node and spread in a planar fashion to induce anterior neural ectoderm. Second, the anterior translocation of organizer-derived cells shifts the source of neuralizing factors anteriorly, where the relative concentration of BMP-4-antagonists is thus elevated, and the medial part of the prospective forebrain becomes competent to respond to ventralizing factors. Third, the forebrain anlage is ventralized by signals including Sonic Hedgehog, thereby creating a new identity, the prospective hypothalamus, which splits the eye anlage into two lateral domains.     

Some electron microscope observations of longitudinal splitting from surface flaws in Kevlar 29

Journal of Materials Science Letters -     

Prophylactic treatment of migraine with gamma-linolenic and alpha-linolenic acids

Polyunsaturated fatty acids (PUFA) were administered to 168 patients over a period of 6 months in an open-label uncontrolled study. In 129 patients available for study, 86% experienced reduction in severity, frequency and duration of migraine attacks, 22% became free of migraine and more than 90% had reduced nausea and vomiting. Self-medication changed to simple analgesics in the majority except in 14% of patients without improvement.