Evaluation of DNA enzymes targeted against the RNA component of human telomerase

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is currently no effective cure. Consequently, developing new therapies and elucidating effective targets is crucial for this fatal disease. In recent years, DNA enzymes, deoxyribonucleic acid molecules with enzymatic activity, have emerged. In the same manner as ribozymes, DNA enzymes are able to effect cleavage of RNA in a sequence-specific manner, and operate with catalytic efficiency. In this study, two DNA enzymes were designed to target the template region of human telomerase RNA (hTR), utilising the 10-23 and 8-17 catalytic motifs elucidated by Santoro and Joyce (1997). Telomerase is an RNA-dependent DNA polymerase, which stabilises telomere lengths by adding hexameric repeats (TTAGGG in humans) to chromosome termini, thus preventing the telomere shortening that usually occurs during mitotic cell division. Telomerase activity, whilst absent in normal somatic tissues, is present in almost 90% of all tumours. Thus, there is speculation that telomerase may be the much sought universal target for therapeutic intervention in cancer. In vitro cleavage assays showed both DNA enzymes to be catalytically competent. Unmodified phosphodiester (PO) backbone DNA enzymes were rapidly degraded in the presence of serum, with a half-life of 10 minutes. The common approach of introducing phosphorothioate (PS) linkages was used in an effort to overcome this instability. As a result of concurrent activity and stability studies on the DNA enzymes with various numbers of PS linkages, the DNA enzymes with a PO core and PS arms were chosen for use in further cell work. The cleavage activity of both was shown to be specific and affected by temperature, pH, MgCI2 concentration and enzyme concentration. Both DNA enzyme motifs reduced telomerase activity in cell lysates, as assessed by the telomerase repeat amplification protocol (TRAP) with an IC50 of 100nM. DNA enzymes being polyanionic molecules do not readily cross biological barriers. Cellular association of naked DNA enzyme was inefficient at less than 2%. Cellular delivery of the DNA enzymes was effectively improved using commercial cationic lipid formulations. However, the lipid-mediated delivery of DNA enzymes to U87-MG cells over a 4-hour period did not significantly inhibit cell proliferation compared to controls. This is possibly due to an expected lag period between the inhibition of telomere maintenance and cell death. Therefore, biodegradable polymer microspheres were investigated as a potential delivery option for prolonged and sustained delivery. In vitro release profiles showed that after an initial burst, sustained release of DNA enzymes was observed over 35 days. Finally, the efficacy and specificity of the DNA enzymes were demonstrated in a luciferase based reporter assay. Specific inhibition of luciferase expression was displayed at 10nM. Thus DNA enzymes have potential against endogenous cellular targets.

The dawn of the RNA World:toward functional complexity through ligation of random RNA oligomers

A main unsolved problem in the RNA World scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers obtained by random polymerization on mineral surfaces. A number of computational studies have shown that the structural repertoire yielded by that process is dominated by topologically simple structures, notably hairpin-like ones. A fraction of these could display RNA ligase activity and catalyze the assembly of larger, eventually functional RNA molecules retaining their previous modular structure: molecular complexity increases but template replication is absent. This allows us to build up a stepwise model of ligation- based, modular evolution that could pave the way to the emergence of a ribozyme with RNA replicase activity, step at which information-driven Darwinian evolution would be triggered. Copyright © 2009 RNA Society.

The opportunity recognition process:the mechanisms by which entrepreneurial opportunities are recognised and the role played by social networks in this process

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The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Transporter associated with antigen processing preselection of peptides binding to the MHC:a bioinformatic evaluation

TAP is responsible for the transit of peptides from the cytosol to the lumen of the endoplasmic reticulum. In an immunological context, this event is followed by the binding of peptides to MHC molecules before export to the cell surface and recognition by T cells. Because TAP transport precedes MHC binding, TAP preferences may make a significant contribution to epitope selection. To assess the impact of this preselection, we have developed a scoring function for TAP affinity prediction using the additive method, have used it to analyze and extend the TAP binding motif, and have evaluated how well this model acts as a preselection step in predicting MHC binding peptides. To distinguish between MHC alleles that are exclusively dependent on TAP and those exhibiting only a partial dependence on TAP, two sets of MHC binding peptides were examined: HLA-A*0201 was selected as a representative of partially TAP-dependent HLA alleles, and HLA-A*0301 represented fully TAP-dependent HLA alleles. TAP preselection has a greater impact on TAP-dependent alleles than on TAP-independent alleles. The reduction in the number of nonbinders varied from 10% (TAP-independent) to 33% (TAP-dependent), suggesting that TAP preselection is an important component in the successful in silico prediction of T cell epitopes.

A model for the modular evolution of rna addressing open questions on the origin of life

A main unsolved problem in the RNA world scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers generated by random polymerization of ribonucleotides (Joyce and Orgel 2006). Current estimates establish a minimum size about 165 nt long for such a ribozyme (Johnston et al. 2001), a length three to four times that of the longest RNA oligomers obtained by random polymerization on clay mineral surfaces (Huang and Ferris 2003, 2006). To overcome this gap, we have developed a stepwise model of ligation-based, modular evolution of RNA (Briones et al. 2009) whose main conceptual steps are summarized in Figure 1. This scenario has two main advantages with respect to previous hypotheses put forward for the origin of the RNA world: i) short RNA....

Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

A comparative evaluation of term recognition algorithms

Automatic Term Recognition (ATR) is a fundamental processing step preceding more complex tasks such as semantic search and ontology learning. From a large number of methodologies available in the literature only a few are able to handle both single and multi-word terms. In this paper we present a comparison of five such algorithms and propose a combined approach using a voting mechanism. We evaluated the six approaches using two different corpora and show how the voting algorithm performs best on one corpus (a collection of texts from Wikipedia) and less well using the Genia corpus (a standard life science corpus). This indicates that choice and design of corpus has a major impact on the evaluation of term recognition algorithms. Our experiments also showed that single-word terms can be equally important and occupy a fairly large proportion in certain domains. As a result, algorithms that ignore single-word terms may cause problems to tasks built on top of ATR. Effective ATR systems also need to take into account both the unstructured text and the structured aspects and this means information extraction techniques need to be integrated into the term recognition process.

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.

Structured engagement in the extended enterprise

Discusses the necessity for the conscious recognition of the phenomenon known as the extended enterprise; this demands that product, process and supply chain design are all considered simultaneously. Structure must be given to the extended enterprise in order to understand and manage it efficaciously. The authors discuss multiple perspectives for doing this, and employ the notions of “3-dimensional concurrent engineering” and “holonic thinking” for conceiving what the structure may look like. Describes a current “action research” project that is investigating potential lead-time reductions within an extended enterprise’s product introduction process. This aims to produce process visualisations, a framework for structuring and sychronising phases and stage-gates within the extended enterprise, and a new simulation tool which will provide a synthetic distributed hypermedia network. These deliverables will be used to play strategic “games” to explore problem issues within the product introduction process that belongs to the extended enterprise, develop teamwork across autonomous companies, and ultimately, contribute to the design of future extended enterprise supply chains.

The de-gaying and re-gaying of AIDS:contested homophobias in lesbian and gay awareness training

From the first recognition of AIDS as a disease, it was publicly conceptualized as a 'gay plague'. In response, health education and diversity training sought to counter this association claiming that AIDS is an 'equal opportunity' virus - that it can affect anyone. In this article, we analyse talk about HIV/AIDS within a data corpus of 13 tape-recorded lesbian and gay awareness training sessions. Counter to the way in which interactions are described in the lesbian and gay awareness training literature, we found that it was trainees, rather than trainers, who pursued discussions about HIV/AIDS, and who did so in order to claim the 'de-gaying' of AIDS, which they treated as representing a 'non-prejudiced' position. By contrast, and in response to trainees' insistence on de-gaying AIDS, trainers were 're-gaying' AIDS. Our analysis highlights that in these sessions - designed explicitly to counter homophobic attitudes - apparently 'factual' claims and counter-claims about infection rates and risk groups are underpinned by essentially contested definitions of what constitutes a 'homophobic' attitude. We conclude by pointing to the value of detailed analysis of talk-in-interaction for understanding professional practices, and suggest strategies for improving the pedagogic value of training. Copyright © 2005 SAGE Publications.

Facial emotion recognition and alexithymia in adults with somatoform disorders

The primary aim of this study was to investigate facial emotion recognition (FER) in patients with somatoform disorders (SFD). Also of interest was the extent to which concurrent alexithymia contributed to any changes in emotion recognition accuracy. Twenty patients with SFD and 20 healthy, age, sex and education matched, controls were assessed with the Facially Expressed Emotion Labelling Test of FER and the 26-item Toronto Alexithymia Scale. Patients withSFD exhibited elevated alexithymia symptoms relative to healthy controls.Patients with SFD also recognized significantly fewer emotional expressions than did the healthy controls. However, the group difference in emotion recognition accuracy became nonsignificant once the influence of alexithymia was controlled for statistically. This suggests that the deficit in FER observed in the patients with SFD was most likely a consequence of concurrent alexithymia. It should be noted that neither depression nor anxiety was significantly related to emotion recognition accuracy, suggesting that these variables did not contribute the emotion recognition deficit. Impaired FER observed in the patients with SFD could plausibly have a negative influence on these individuals’ social functioning.

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

Developmental trajectories of part-based and configural object recognition in adolescence.

Three experiments assessed the development of children's part and configural (part-relational) processing in object recognition during adolescence. In total, 312 school children aged 7-16 years and 80 adults were tested in 3-alternative forced choice (3-AFC) tasks. They judged the correct appearance of upright and inverted presented familiar animals, artifacts, and newly learned multipart objects, which had been manipulated either in terms of individual parts or part relations. Manipulation of part relations was constrained to either metric (animals, artifacts, and multipart objects) or categorical (multipart objects only) changes. For animals and artifacts, even the youngest children were close to adult levels for the correct recognition of an individual part change. By contrast, it was not until 11-12 years of age that they achieved similar levels of performance with regard to altered metric part relations. For the newly learned multipart objects, performance was equivalent throughout the tested age range for upright presented stimuli in the case of categorical part-specific and part-relational changes. In the case of metric manipulations, the results confirmed the data pattern observed for animals and artifacts. Together, the results provide converging evidence, with studies of face recognition, for a surprisingly late consolidation of configural-metric relative to part-based object recognition.

Innate recognition of apoptotic cells:novel apoptotic cell-associated molecular patterns revealed by crossreactivity of anti-LPS antibodies

Cells dying by apoptosis are normally cleared by phagocytes through mechanisms that can suppress inflammation and immunity. Molecules of the innate immune system, the pattern recognition receptors (PRRs), are able to interact not only with conserved structures on microbes (pathogen-associated molecular patterns, PAMPs) but also with ligands displayed by apoptotic cells. We reasoned that PRRs might therefore interact with structures on apoptotic cells-apoptotic cell-associated molecular patterns (ACAMPs)-that are analogous to PAMPs. Here we show that certain monoclonal antibodies raised against the prototypic PAMP, lipopolysaccharide (LPS), can crossreact with apoptotic cells. We demonstrate that one such antibody interacts with a constitutively expressed intracellular protein, laminin-binding protein, which translocates to the cell surface during apoptosis and can interact with cells expressing the prototypic PRR, mCD14 as well as with CD14-negative cells. Anti-LPS cross reactive epitopes on apoptotic cells colocalised with annexin V-and C1q-binding sites on vesicular regions of apoptotic cell surfaces and were released associated with apoptotic cell-derived microvesicles (MVs). These results confirm that apoptotic cells and microbes can interact with the immune system through common elements and suggest that anti-PAMP antibodies could be used strategically to characterise novel ACAMPs associated not only with apoptotic cells but also with derived MVs. © 2013 Macmillan Publishers Limited All rights reserved.