Nitric Oxide Synthase in Confined Environments;Detection and Quantification of Nitric Oxide Released from Cells and Modified Liposomes Using a Sensitive Metal Catalyst-Pedot Modified Carbon Fiber Electrode

Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes that include neuronal signaling, immune response, and inflammatory response. NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. A deficiency in NO plays a role in hypertension, hyperglycaemia, and arteriosclerosis, among other pathological states. Conversely, increased NO levels contribute to arthritis, septic shock, and hypotention. Therefore, measuring and quantifying NO production in biological systems and matrices may be vital in elucidating physiological and pathological processes. The goal of this work is to develop an ultra-sensitive and selective electrochemical sensor taking advantage of NO-sulfur chemistry. In particular, electropolymerizing 3,4-ethylenedioxythiophene (EDOT) monomers on the surface of our electrodes yield a suitable sulfur-based polymer PEDOT to be used as an affinity matrix for NO sensing. In other work, we have shown that the ruthenium (Ru) mediates the catalytic oxidation of NO. In this work, we tried to achieve improved sensitivity by combining both Ru nanoparticles and PEDOT using the layer-by-layer (LBL) modification method. Further, to eliminate interferences the Ru-PEDOT-Ru modified carbon fiber was coated with a nafion layer, which acts as an anionic filter. We used our NO-sensor to accurately monitor NO release from mouse embryonic fibroblast cells as well as isolated single human umbilical vein endothelial cells. A second part of this work focused on testing the performance of our sensors in characterizing NO release from liposomes with confined NOS enzyme. Liposomes are spherical, closed, self-assembled phospholipids, which enclose part of the surrounding solvent in their interior. Liposomes can enclose an aqueous medium separate from the external aqueous medium. Therefore, liposomes can be used as carriers of enzymes (NOS in this case) without negative impact on the molecular

Nitric Oxide Synthase in Confined Environments;Detection and Quantification of Nitric Oxide Released from Cells and Modified Liposomes Using a Sensitive Metal Catalyst-Pedot Modified Carbon Fiber Electrode

Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes that include neuronal signaling, immune response, and inflammatory response. NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. A deficiency in NO plays a role in hypertension, hyperglycaemia, and arteriosclerosis, among other pathological states. Conversely, increased NO levels contribute to arthritis, septic shock, and hypotention. Therefore, measuring and quantifying NO production in biological systems and matrices may be vital in elucidating physiological and pathological processes. The goal of this work is to develop an ultra-sensitive and selective electrochemical sensor taking advantage of NO-sulfur chemistry. In particular, electropolymerizing 3,4-ethylenedioxythiophene (EDOT) monomers on the surface of our electrodes yield a suitable sulfur-based polymer PEDOT to be used as an affinity matrix for NO sensing. In other work, we have shown that the ruthenium (Ru) mediates the catalytic oxidation of NO. In this work, we tried to achieve improved sensitivity by combining both Ru nanoparticles and PEDOT using the layer-by-layer (LBL) modification method. Further, to eliminate interferences the Ru-PEDOT-Ru modified carbon fiber was coated with a nafion layer, which acts as an anionic filter. We used our NO-sensor to accurately monitor NO release from mouse embryonic fibroblast cells as well as isolated single human umbilical vein endothelial cells. A second part of this work focused on testing the performance of our sensors in characterizing NO release from liposomes with confined NOS enzyme. Liposomes are spherical, closed, self-assembled phospholipids, which enclose part of the surrounding solvent in their interior. Liposomes can enclose an aqueous medium separate from the external aqueous medium. Therefore, liposomes can be used as carriers of enzymes (NOS in this case) without negative impact on the molecular

Nitric Oxide Synthase in Confined Environments;Detection and Quantification of Nitric Oxide Released from Cells and Modified Liposomes Using a Sensitive Metal Catalyst-Pedot Modified Carbon Fiber Electrode

Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes that include neuronal signaling, immune response, and inflammatory response. NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. A deficiency in NO plays a role in hypertension, hyperglycaemia, and arteriosclerosis, among other pathological states. Conversely, increased NO levels contribute to arthritis, septic shock, and hypotention. Therefore, measuring and quantifying NO production in biological systems and matrices may be vital in elucidating physiological and pathological processes. The goal of this work is to develop an ultra-sensitive and selective electrochemical sensor taking advantage of NO-sulfur chemistry. In particular, electropolymerizing 3,4-ethylenedioxythiophene (EDOT) monomers on the surface of our electrodes yield a suitable sulfur-based polymer PEDOT to be used as an affinity matrix for NO sensing. In other work, we have shown that the ruthenium (Ru) mediates the catalytic oxidation of NO. In this work, we tried to achieve improved sensitivity by combining both Ru nanoparticles and PEDOT using the layer-by-layer (LBL) modification method. Further, to eliminate interferences the Ru-PEDOT-Ru modified carbon fiber was coated with a nafion layer, which acts as an anionic filter. We used our NO-sensor to accurately monitor NO release from mouse embryonic fibroblast cells as well as isolated single human umbilical vein endothelial cells. A second part of this work focused on testing the performance of our sensors in characterizing NO release from liposomes with confined NOS enzyme. Liposomes are spherical, closed, self-assembled phospholipids, which enclose part of the surrounding solvent in their interior. Liposomes can enclose an aqueous medium separate from the external aqueous medium. Therefore, liposomes can be used as carriers of enzymes (NOS in this case) without negative impact on the molecular

Historical context of profesional ideology and tension and strain in the accounting profession

A growing literature points to a crisis of confidence in the accounting profession and a lack of commitment by its members to the professional ideology. In this paper the approach developed by MacIntyre is used to place professional ideology in an historical context. The paper argues that the tension and strain in the profession can be related to the changing character of both the contemporary society and professional ideology itself. It concludes by highlighting the need for the profession to develop an ideology to which its members as well as society can relate

Evaluation and Application of Stationary Phase Selectivity for Drug Analysis

Despite the wide range of HPLC stationary phases available for reversed-phase high-performance liquid chromatography (RP-HPLC) and the in-depth studies using probes to highlight differences between them, there is very little in the way of stationary phases which offer selectivity that is substantially different from that offered by the very commonly used alkyl-silicas. Therefore, the primary aim of the research programme was to explore and try to exploit LC stationary phases which offered genuinely different selectivity to alkyl-silicas for typical drug applications. Chiral stationary phases (CSP) potentially had different selectivity and in this context a secondary aim was to explore aspects of the enantioselectivity of CSP as well as their chemical selectivity. Claims of orthogonal selectivity had been made for pentafluorophenyl (PFP) phases and phases exhibiting the hydrophilic interaction liquid chromatography (HILIC) mode. However, the Ultra PFP phase was found to be very similar in selectivity to ACE 5 C18 for both amitriptyline and acemetacin related compounds. The ZIC-HILIC phase was shown to behave as a reversed-phase material at high aqueous content in the mobile phase. There was some indication of selectivity orthogonal to that of ACE 5 C18 with low aqueous content in the mobile phase but this occurred at low retention and with mobile phases unsuitable for use with C18 phases in coupled (column or phase) systems. Nonetheless the work carried out shed more light on the mechanisms taking place in the HILIC mode which is currently attracting so much interest. Also it was possible to put ZIC-HILIC to good use for polar plant metabolites and other applications. Chiral stationary phases (CSP) also offered the prospect of selectivity orthogonal to that of C18 phases. Given the proliferation of such phases though and the fact that it would be useful to use CSP that gave chiral separations for a broad spectrum of compound classes as well as giving orthogonal separations between different compounds, it was decided to carry out comparative studies of CSP classes in order to identify any redundancies and to seek out CSP that were complementary to one another. The Regis Whelk-O1 CSP was shown to be much superior to other higher-generation Pirkle-concept CSP such as DACH-DNB and ULMO. Also it was shown to be complementary to the Chiralcel OD derivatised ii polysaccharide CSP and that both had something to offer alongside the widely used Chiralpak AD derivatised polysaccharide CSP. It was also found that a series of Chiralcel OD clones were virtually identical to Chiralcel OD and similarly for Chiralpak AD clones. Chiralpak IA, an immobilised version of Chiralpak AD, was not markedly less enantioselective than Chiralpak AD. Chiralcel OJ was less enantioselective than Chiralpak AD but the gap in performance was not as wide as between Whelk-O1 and the other Pirkle-concept CSP. The information gathered during these studies should prove to be of enormous value for further work in chiral LC method development screening. Before embarking on applications work utilising the stationary phase selectivity that had been found, a study was carried out on the effectiveness of the high efficiencies obtainable with short run times through ultra-performance liquid chromatography (UPLC). It was found that, for a range of pharmaceutical applications, that it was still necessary in each case to adjust selectivity before increasing speed through working at higher temperatures with faster flow rates. In the course of this work some exceptionally high speed separations for example for paroxetine and related substances, benzodiazepines and flurbiprofen and related substances, were developed. With respect to the evaluation of CSP as orthogonal phases to alkyl silicas under reversed-phase conditions, the Whelk-O1 CSP showed promise. However on closer inspection it was found that the Whelk-O1 CSP had very similar selectivity to the alkyl silica phase, ACE 5 C18, and deviation from this only occurred in instances when there was interaction with the chiral recognition site to give a separation of enantiomers. This prompted the notion that, rather than using Whelk- O1 in a coupled column system with ACE 5 C18, it could be used on its own for the separation of both trace enantiomer and all other related substances. This was shown to be possible using (S)-naproxen, laevokalim and (S)-flurbiprofen as illustrative examples. The evaluation of the enantioselectivity of CSP led to an optimised resolution (suitable for scaling up for preparative work) of the enantiomers of the former ‘legal-high’ drug, mephedrone, on Whelk-O1 under normal phase conditions. It was also shown that the infrequently used Chiralcel OJ derivatised polysaccharide iii CSP was ideal for developing an assay to determine trace amounts of (R)-nicotine in (S)-nicotine. Overall, the information obtained on stationary phase selectivity and retentivity through evaluation and application will be of great value in HPLC and UHPLC column selection and also selection of orthogonal phases for coupled column systems but, ultimately, moving forward, most value may be in aiding the design of two-dimensional LC systems for complex mixture analysis. This would particularly apply to the use of CSP with reversed-phase eluents in achiral-chiral systems

Benchmarking network propagation methods for disease gene identification

In-silico identification of potential target genes for disease is an essential aspect of drug target discovery. Recent studies suggest that successful targets can be found through by leveraging genetic, genomic and protein interaction information. Here, we systematically tested the ability of 12 varied algorithms, based on network propagation, to identify genes that have been targeted by any drug, on gene-disease data from 22 common non-cancerous diseases in OpenTargets. We considered two biological networks, six performance metrics and compared two types of input gene-disease association scores. The impact of the design factors in performance was quantified through additive explanatory models. Standard cross-validation led to over-optimistic performance estimates due to the presence of protein complexes. In order to obtain realistic estimates, we introduced two novel protein complex-aware cross-validation schemes. When seeding biological networks with known drug targets, machine learning and diffusion-based methods found around 2-4 true targets within the top 20 suggestions. Seeding the networks with genes associated to disease by genetics decreased performance below 1 true hit on average. The use of a larger network, although noisier, improved overall performance. We conclude that diffusion-based prioritisers and machine learning applied to diffusion-based features are suited for drug discovery in practice and improve over simpler neighbour-voting methods. We also demonstrate the large impact of choosing an adequate validation strategy and the definition of seed disease genesPeer ReviewedPostprint (published version

Review of EEG-based pattern classification frameworks for dyslexia

Dyslexia is a disability that causes difficulties in reading and writing despite average intelligence. This hidden disability often goes undetected since dyslexics are normal and healthy in every other way. Electroencephalography (EEG) is one of the upcoming methods being researched for identifying unique brain activation patterns in dyslexics. The aims of this paper are to examine pros and cons of existing EEG-based pattern classification frameworks for dyslexia and recommend optimisations through the findings to assist future research. A critical analysis of the literature is conducted focusing on each framework’s (1) data collection, (2) pre-processing, (3) analysis and (4) classification methods. A wide range of inputs as well as classification approaches has been experimented for the improvement in EEG-based pattern classification frameworks. It was uncovered that incorporating reading- and writing-related tasks to experiments used in data collection may help improve these frameworks instead of using only simple tasks, and those unwanted artefacts caused by body movements in the EEG signals during reading and writing activities could be minimised using artefact subspace reconstruction. Further, support vector machine is identified as a promising classifier to be used in EEG-based pattern classification frameworks for dyslexia