Peptide nucleic acid conjugates regulate gene expression in vitro: Mode of action and new strategies for improved cellular uptake

RNA G-quadruplexes (G4) are an important class of nucleic acid secondary structures that are involved in mRNA translation, alternative splicing, localisation and 3’-end processing. Putative RNA G4 forming sequences have been identified in the regulatory regions of many disease-related genes, in particular oncogenes, and as a result have been of increasing interest as therapeutic targets for chemical intervention. G4-binding small molecule ligands and antisense oligonucleotides have both been used to effectively regulate translation. However, the therapeutic potential of drugs targeting RNA G4 structures is limited by a lack of specificity in the presence of alternative RNA secondary structures (including other G4s). Herein, the efficacy of a new generation of “sequence + structure” specific RNA G4 ligands is tested against the well characterised NRAS G4, chosen as a model system. Ligands were designed to target both (i) G4-specific structural features using a flat aromatic and cationic NDI platform and (ii) the single-stranded G4 flanking regions using short complementary peptide nucleic acid (PNA) sequences. Using an in vitro translation assay these PNA-small molecule conjugates were shown to inhibit translation with a significantly lower IC50 than the PNA or NDI alone. However, evidence suggested that the observed effect was mainly non-specific, which we suggest is due to electrostatic interactions mediated by the positively charged lysine residues added to the PNA-conjugates to improve solubility. In addition, a new 5’UTR G4 in the mRNA of the Aurora A kinase gene has been identified and characterised. Therapeutic PNA oligomers, built around a neutral peptide backbone instead of the negatively-charged oligonucleotide’s sugar-phosphate backbone, offer a number of advantages when compared to natural oligonucleotides, including resistance to proteases and higher binding affinity and specificity for complementary DNA (or RNA) sequences. However, PNA oligomers have very poor membrane permeability, which means that PNA conjugation is likely to significantly decrease their bioavailability. Conjugation of molecular transporters, such as cell penetrating peptides (or peptoids), is currently the most viable method to improve cellular uptake of therapeutic PNA oligomers. Here we report on the development of an in vitro assay and present initial results showing that sonoporation (formation of small pores in cell membranes by combined use of ultrasound and microbubbles) can be used to increase the cellular uptake of PNAs without the need for pre-conjugation to cell-penetrating peptides.Open Acces

Bid/ask Spreads And The Costs Of Making Markets

The purpose of this thesis is to test the received explanations of the determinants of bid/ask spreads for common equity securities. This thesis employs trade-to-trade data for twenty-four securities listed on the Toronto Stock Exchange (TSE) over the period from August, 1988 through December, 1992. The literature is extended by simultaneously modelling the cost components (inventory, asymmetric information, fixed, and search costs) and volatility of realized bid/ask spreads using an exponential autoregressive conditional heteroscedasticity (EARCH) framework. Because certain small trades on the TSE are auto-filled, registered traders do not always make a decision on trade involvement--some trades are non-discretionary. The theoretical model of spreads used in this thesis extends Glosten and Harris\u27 (52) (GH) model by distinguishing between non-discretionary (auto-filled) and discretionary trades to allow testing for differences in cost structures. Determination of the relative contribution of the various cost components is also based on GH.;The contributions of this thesis are threefold: (1) the costs of trading are dependent on the trade type--auto-filled versus discretionary; (2) the volatility of realized spreads is modelled as an exponential autoregressive conditionally heteroscedastic (EARCH) process which is a function of information flow; and (3) richer, more exact intra-daily data is used to eliminate problems currently recognized in the literature. The primary conclusions are: (1) for non-discretionary trades, the average cost per share per trade is {dollar}0.0683 of which 96\% is due to fixed costs and 4\% is an asymmetric information cost; (2)~for discretionary trades, the average cost is \{dollar}0.0273 of which 76% is fixed and 24% is information: and (3) search (liquidity) and inventory costs are not statistically different from 0. These results are consistent over three different sub-periods

WFIRST Coronagraph Technology Requirements: Status Update and Systems Engineering Approach

The coronagraphic instrument (CGI) on the Wide-Field Infrared Survey Telescope (WFIRST) will demonstrate technologies and methods for high-contrast direct imaging and spectroscopy of exoplanet systems in reflected light, including polarimetry of circumstellar disks. The WFIRST management and CGI engineering and science investigation teams have developed requirements for the instrument, motivated by the objectives and technology development needs of potential future flagship exoplanet characterization missions such as the NASA Habitable Exoplanet Imaging Mission (HabEx) and the Large UV/Optical/IR Surveyor (LUVOIR). The requirements have been refined to support recommendations from the WFIRST Independent External Technical/Management/Cost Review (WIETR) that the WFIRST CGI be classified as a technology demonstration instrument instead of a science instrument. This paper provides a description of how the CGI requirements flow from the top of the overall WFIRST mission structure through the Level 2 requirements, where the focus here is on capturing the detailed context and rationales for the CGI Level 2 requirements. The WFIRST requirements flow starts with the top Program Level Requirements Appendix (PLRA), which contains both high-level mission objectives as well as the CGI-specific baseline technical and data requirements (BTR and BDR, respectively)... We also present the process and collaborative tools used in the L2 requirements development and management, including the collection and organization of science inputs, an open-source approach to managing the requirements database, and automating documentation. The tools created for the CGI L2 requirements have the potential to improve the design and planning of other projects, streamlining requirement management and maintenance. [Abstract Abbreviated]Comment: 16 pages, 4 figure

Mitochondrial defects in acute multiple sclerosis lesions

Multiple sclerosis is a chronic inflammatory disease, which leads to focal plaques of demyelination and tissue injury in the CNS. The structural and immunopathological patterns of demyelination suggest that different immune mechanisms may be involved in tissue damage. In a subtype of lesions, which are mainly found in patients with acute fulminant multiple sclerosis with Balo's type concentric sclerosis and in a subset of early relapsing remitting multiple sclerosis, the initial myelin changes closely resemble those seen in white matter stroke (WMS), suggesting a hypoxia-like tissue injury. Since mitochondrial injury may be involved in the pathogenesis of such lesions, we analysed a number of mitochondrial respiratory chain proteins in active lesions from acute multiple sclerosis and from WMS using immunohistochemistry. Functionally important defects of mitochondrial respiratory chain complex IV [cytochrome c oxidase (COX)] including its catalytic component (COX-I) are present in Pattern III but not in Pattern II multiple sclerosis lesions. The lack of immunohistochemically detected COX-I is apparent in oligodendrocytes, hypertrophied astrocytes and axons, but not in microglia. The profile of immunohistochemically detected mitochondrial respiratory chain complex subunits differs between multiple sclerosis and WMS. The findings suggest that hypoxia-like tissue injury in Pattern III multiple sclerosis lesions may be due to mitochondrial impairment

The role of the UPS in cystic fibrosis

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the ΔF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of ‘corrector’ drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients

The genome sequence of the hornet moth, Sesia apiformis (Clerck, 1759)

We present a genome assembly from an individual male Sesia apiformis (the Hornet Moth; Arthropoda; Insecta; Lepidoptera; Sesiidae). The genome sequence is 546.8 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,358 protein coding genes

Far-field deformation resulting from rheologic differences interacting with tectonic stresses: an example from the Pacific/Australian plate boundary in Southern New Zealand

The Miocene in Southern New Zealand was dominated by strike-slip tectonics. Stratigraphic evidence from this time attests to two zones of subsidence in the south: (a) a middle Cenozoic pull-apart basin and (b) a regionally extensive subsiding lake complex, which developed east and distal to the developing plate boundary structure. The lake overlay a block of crust with a significantly weak mid-crustal section and we pose the question: can rheological transitions at an angle to a plate boundary produce distal subsidence and/or uplift? We use stratigraphic, structural and geophysical observations from Southern New Zealand to constrain three-dimensional numerical models for a variety of boundary conditions and rheological scenarios. We show that coincident subsidence and uplift can result from purely strike-slip boundary conditions interacting with a transition from strong to weak to strong mid-crustal rheology. The resulting pattern of vertical displacement is a function of the symmetry or asymmetry of the boundary conditions and the extent and orientation of the rheological transitions. For the Southern New Zealand case study, subsidence rates of ~0.1 mm/yr are predicted for a relative plate motion of 25 mm/yr, leading to ~500 m of subsidence over a 5 Ma time period, comparable to the thickness of preserved lacustrine sediments

Denervation Causes Fiber Atrophy and Myosin Heavy Chain Co-Expression in Senescent Skeletal Muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm−1) and SEN (469±13 MNs×mm−1). Nav1.5 positive fibers (1548±70 μm2) were 35% smaller than Nav1.5 negative fibers (2367±78 μm2; P<0.05) in SEN muscle, whereas Nav1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm2; P<0.05) where no Nav1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy