Endo-Porter–Mediated Delivery of Phosphorodiamidate Morpholino Oligos (PMOs) in Erythrocyte Suspension Cultures from Cope\u27s Gray Treefrog Hyla Chrysoscelis

Cope\u27s gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)–mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in \u3e65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry)

Date of Receiving: to be completed by the Editor Date of Acceptance: to be completed by the Editor Temporal Redundancy Based Encoding Technique for Peak Power and Delay Reduction of On-Chip Buses

Abstract —Power consumption and delay are two of the most important constraints in current-day on-chip bus design. The two major sources of dynamic power dissipation on a bus are the self capacitance and the coupling capacitance. As technology scales, the interconnect resistance increases due to shrinking wire-width. At the same time, spacing between the interconnects decreases resulting in an increase in the coupling capacitance. This, in turn, leads to stronger crosstalk effects between the interconnects. In Deep Sub-Micron technology the coupling capacitance exceeds the self capacitance, which, in turn, cause more power consumption and delay on the bus. Recently, the interest has also shifted to minimizing peak power dissipation. The reason being that higher peak power leads to an undesired increase in switching noise, metal electromigration problems and operationinduced variations due to non-uniform temperature on the die. Thus, minimizing power consumption and delay are the most important design objectives for on-chip buses. Several bus encoding schemes have been proposed in the literature for reducing crosstalk. Most of these encoding techniques use spatial redundancy that requires additional transmission wires on the bus. In this paper, a new temporal encoding scheme is proposed

Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10 kb C-to-T splicing mutation

Background Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene. Methods We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2′-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele. Results A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2′-Methoxy Ethyl modification (2′MOE). Conclusion The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development

Universal Matrix Insertion Grammars with Small Size

International audienc

Sm-like protein Hfq: Location of the ATP-binding site and the effect of ATP on Hfq–RNA complexes

Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq–RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins