Single stranded fully Modified-Phosphorothioate oligonucleotides can induce structured nuclear inclusions, alter nuclear protein localization and disturb the transcriptome In Vitro

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects

Modulation control and spectral shaping of optical fiber supercontinuum generation in the picosecond regime

Numerical simulations are used to study how fiber supercontinuum generation seeded by picosecond pulses can be actively controlled through the use of input pulse modulation. By carrying out multiple simulations in the presence of noise, we show how tailored supercontinuum Spectra with increased bandwidth and improved stability can be generated using an input envelope modulation of appropriate frequency and depth. The results are discussed in terms of the non-linear propagation dynamics and pump depletion.Comment: Aspects of this work were presented in Paper ThJ2 at OECC/ACOFT 2008, Sydney Australia 7-10 July (2008). Journal paper submitted for publication 30 July 200

Targeted exon skipping to correct exon duplications in the dystrophin gene

Duchenne muscular dystrophy is a severe muscle-wasting disease caused by mutations in the dystrophin gene that ablate functional protein expression. Although exonic deletions are the most common Duchenne muscular dystrophy lesion, duplications account for 10–15% of reported disease-causing mutations, and exon 2 is the most commonly duplicated exon. Here, we describe the in vitro evaluation of phosphorodiamidate morpholino oligomers coupled to a cell-penetrating peptide and 2′-O-methyl phosphorothioate oligonucleotides, using three distinct strategies to reframe the dystrophin transcript in patient cells carrying an exon 2 duplication. Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide proving the most effective. Differences in exon 2 excision efficiency between normal and exon 2 duplication cells, were apparent, indicating that exon context influences oligomer-induced splice switching. Skipping of a single copy of exon 2 was induced in the cells carrying an exon 2 duplication, the simplest strategy to restore the reading frame and generate a normal dystrophin transcript. In contrast, multiexon skipping of exons 2–7 to generate a Becker muscular dystrophy-like dystrophin transcript was more challenging and could only be induced efficiently with the phosphorodiamidate morpholino oligomer chemistry

Undiliuted Isotopic Measurements of Meteoritic Nano-Oxides Through Atom Probe Tomography

We present results for the first atom probe tomography study of a meteoritic chromite

Mechanical Properties and Nanocrystallization Behavior of Al-Ni-La Alloys

Rapidly solidified Al89Ni6La5 ribbons were obtained by induction melting and ejecting the melt onto a rotating Cu wheel in an Ar atmosphere. The ribbons were investigated by differential scanning calorimetry (DSC), X-ray diffractometry (XRD), transmission electron microscopy (TEM), microindentation, and nanoindentation techniques. The XRD and TEM studies revealed that the ribbon was fully amorphous; however, DSC did not show any glass transition. The alloy undergoes two stages of crystallization. The growth of fcc-Al is responsible for the first stage, and precipitation of Al3Ni and Al11La3 is responsible for the second stage of crystallization. Microhardness of ribbons in the as-melt-spun, partially, and fully devitrified conditions was examined and subsequently correlated with evolved microstructure. Significant improvement in hardness was observed, with the progress of primary nanocrystallization, due to the effective barrier to shear band by a hard La-rich shell around the fcc-Al nanocrystals and enrichment of the remaining amorphous matrix by the solute elements. The pile up of materials in the form of semicircular shear bands was observed around all the indentations. During nanoindentation, it was observed that hardness and modulus values were initially increased and then decreased. The reasons for such observation were also discussed. This article is based on a presentation given in the symposium entitled “Mechanical Behavior of Nanostructured Materials,” which occurred during the TMS Spring Meeting in San Francisco, CA, February 15–19, 2009, under the auspices of TMS, the TMS Electronic, Magnetic, and Photonic Materials Division, the TMS Materials Processing and Manufacturing Division, the TMS Structural Materials Division, the TMS Nanomechanical Materials Behavior Committee, the TMS Chemistry and Physics of Materials Committee, and the TMS/ASM Mechanical Behavior of Materials Committee