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.     

Surface leakage currents in SiN/sub x/ passivated AlGaN/GaN HFETs

A novel guarded surface leakage test structure is used to isolate the surface and bulk leakage contributions to gate current in AlGaN/GaN HFETs. Passivation with various recipes of SiN/sub x/ always resulted in the commonly observed increase in gate leakage, which was found to be dominated by bulk leakage through the AlGaN. However, high temperature deposited SiN/sub x/ recipes gave a 1-2 orders reduction in surface leakage, whereas low temperature deposition gave an increase. Gate lag measurements were found to correlate closely with the surface leakage component, giving direct evidence that the key device problem of current slump is associated with current flow at the AlGaN surface.     

Retinoblastoma: clinical and molecular diagnostic aspects

Retinoblastoma, a tumor of the immature retina concerns babies and young infants in particular. They make up for 14% of malignomas in the first years of life. There are two types of retinoblastoma: In the first two alleles of the gene Rb1 must be inactivated sequentially in the same retinoblast cell until this may escape control. In this case the retinoblastoma is always unilateral and unifocal. This is explained by the lower frequency of two mutations in one retinoblast. The other type, however, is inherited: One allele Rb1 is inactivated in all cells of the organism by mutation. The probability that a second mutation arrives in different retinoblasts is thus high. In this case bilateral multifocal tumors develop. Characterization of the Rb1 gene has permitted identification or at least determination of a haplotype in persons at risk. This knowledge is decisive for early recognition of babies at risk and for genetic counselling.     

Microstructure and morphology of amine-cured epoxy coatings before and after outdoor exposures—An AFM study

Atomic force microscopy (AFM) has been used to study the morphology and microstructure of an amine-cured epoxy before and after outdoor exposure. Measurements were made from samples prepared in an essentially CO₂-free, H₂O-free glove box and from samples prepared in ambient conditions. For those prepared in a CO₂-free glove box, AFM imaging was conducted on (1) an unexposed air/coating surface, (2) an unexposed coating bulk, (3) an unexposed coating/substrate interface, and (4) a field exposed air/coating surface. For samples prepared in ambient conditions, only the unexposed air/coating surface was investigated. The same regions of the exposed samples were scanned periodically by the AFM to monitor changes in the surface morphology of the coating as UV exposure progressed. Small angle neutron scattering and Fourier transform infrared spectroscopy (FTIR) studies were performed to verify the microstructure and to follow chemical changes during outdoor exposure, respectively. The results have shown that amine blushing, which occurs only under ambient conditions, had a significant effect on the surface morphology and microstructure of the epoxy. The surface morphology of the samples prepared under CO₂-free, dry conditions was generally smooth and homogeneous. However, the interface and the bulk samples clearly revealed a two-phase structure consisting of bright nodular domains and dark interstitial regions, indicating an inhomogeneous microstructure. Such heterogeneous structure of the bulk was in good agreement with results obtained by small angle neutron scattering of unexposed samples and by AFM phase imaging of the degraded sample surface. The relationship between submicrometer physical changes and molecular chemical degradation is discussed. Presented at the 82nd Annual Meeting of the Federation of Societies for Coatings Technology, October 27–29, 2004, in Chicago, IL.     

Use of laser scanning confocal microscopy for characterizing changes in film thickness and local surface morphology of UV-exposed polymer coatings

Laser scanning confocal microscopy (LSCM) has been used to characterize the changes in film thickness and local surface morphology of polymer coatings during the UV degradation process. With the noninvasive feature of LSCM, one can obtain thickness information directly and nondestructively at various exposure times without destroying the specimens or deriving the thickness values from IR measurement by assuming uniform film ablation. Two acrylic polymer coatings were chosen for the study, and the physical and chemical changes of the two systems at various exposure times were measured and analyzed. Those measurable physical changes caused by UV exposure include film ablation, formation of pits and other surface defects, and increases in surface roughness. It was found in both coatings that changes in measured film thickness by LSCM were not correlated linearly to the predicted thickness loss using the changes in the CH band obtained by the Fourier Transform Infrared (FTIR) spectroscopy measurements in the later degradation stages. This result suggested it was not a uniform film ablation process during the UV degradation. At later stages, where surface deformation became severe, surface roughness and profile information using LSCM were also proven to be useful for analyzing the surface degradation process Presented at the 81st Annual Meeting of the Federation of Societies for Coatings Technology, November 13–14, 2004 in Philadelphia, PA.     

Crystal Chemistry and Phase Transitions in Substituted Pollucites along the CsAlSi2O6-CsTiSi2O6.5 Join: A Powder Synchrotron X-ray Diffractometry Study

The crystal structures for a suite of substituted pollucites with the compositions CsTi _x Al_{1– x} Si₂O_{6+0.5 x} , 0 ≤ x ≤ 1, have been determined from Rietveld analysis of powder synchrotron XRD data. Our results indicate that the pollucite end member (CsAlSi₂O₆) has a tetragonal structure (space group I 4₁/ a ), whereas all other compositions are cubic (space group Ia 3 d ). The increased symmetry for the titanium-substituted structures is presumably due to the incorporation of additional O²⁻ anions (needed for compensating the charge imbalance between Ti⁴⁺ and Al³⁺), which effectively holds open the expanded cubic framework. In situ cooling experiments of the substituted phase CsTi_0.1Al_0.9Si₂O_6.05 reveal a displacive transformation to the tetragonal structure at ∼230 K. This transformation is tricritical in nature and is analogous to the tetragonal-to-cubic transition in pollucite on heating.     

Relationship between chemical degradation and thickness loss of an amine-cured epoxy coating exposed to different UV environments

The relationship between chemical degradation and thickness loss of an unpigmented, non UV-stabilized, crosslinked amine-cured epoxy coating exposed to three UV conditions was investigated. Spin-coated samples having a thickness of approximately 7 μm on an Si substrate were prepared from a stochiometric mixture of a bisphenol A epoxy resin and a tetra-functional amine curing agent. Samples were exposed outdoors and to two accelerated laboratory UV environments. Chemical degradation and thickness loss were measured by transmission Fourier transform infrared spectroscopy (FTIRS) and laser scanning confocal microscopy (LSCM), respectively. In addition, surface roughness and morphological changes were measured by atomic forcemicrosocopy (AFM) and LSCM. Substantial chemical degradation, thickness loss, and morpholocal changes occurred in the exposed films, and the rate of chemical degradation was greater than that due to the thickness loss. This additional chemical loss was attributed to an inhomogeneous degradation process in which nanoscale localized depressions initiate at certain sites on the surface, which then enlarge and deepen with exposure time. The results of this study provide a better understanding of the degradation mechanism and should lead to the development of scientific-based models for predicting the service life of crosslinked amine-cured epoxy coatings. Presented at the 82nd Annual Meeting of the Federation of Societies for Coatings Technology, October 27–29, 2004, in Chicago, IL     

Inverse solution for a biochemical heat source in a porous medium in the presence of natural convection

The inverse problem approach by conjugate gradient with adjoint equations is adapted to the context of a non-isothermal bioprocess, controlled by internal heat generation from microbial oxidation, as could be found in a composting reactor for instance, to determine the heat source from internal temperature measurements. The volumetric heat source is assumed proportional to the rate of consumption of a substrate by a biomass, as described by a Monod model. Computations are performed for Rayleigh numbers equal to 0.25 and 25, for a representative biochemical reaction under typical boundary conditions, for constant and temperature-dependent model parameters. The influence of noisy input data is also considered. It is found that good solutions can be obtained when heat release and diffusion occur over very different time scales. The variation of the model parameters with temperature must be taken into account, but single sensor solutions are possible at relatively small Rayleigh numbers when convection is present.     

Metastable Phase Selection and Partitioning for Zr(1−x)AlxO(2−x/2) Materials Synthesized with Liquid Precursors

Aqueous solutions of zirconium acetate and aluminum nitrate were spray pyrolyzed at 250°C and upquenched to different temperatures to yield metastable solid solutions of composition Zr_{(1− x )}Al_xO_{(2− x /2)}. An amorphous oxide forms first during pyrolysis which subsequently crystallizes as a single phase for x ≤ 0.57 (≤40 mol% Al₂O₃). The crystallization temperature increased with Al₂O₃ content. Electron diffraction, supported by Raman spectroscopy, indicates that the initial phase is tetragonal. At higher temperatures, the initial solid solation partitions to other metastable phases, viz., t -ZrO₂+γ-Al₂O₃, prior to achieving their equilibrium phase assemblage, m -ZrO₂+α-Al₂O₃. Partitioning yields a nanocomposite microstructure with grain sizes of 20–100 nm, compared to the 3 to 5 nm in the initial, single phase. Compositions containing 45 to 50 mol% Al₂O₃ concurrently crystallize and partition. The structure selected during crystallization and the partitioning phenomena are discussed in terms of diffusional constraints during crystallization, which are conceptually similar to those operating during rapid solidification.