Generation of cell lines to complement Adenovirus vectors using recombination-mediated cassette exchange

Background Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression

Quality of Life Improvement with Sublingual Immunotherapy: A Prospective Study of Efficacy

Due to its excellent safety profile, ease of administration, and economic considerations, sublingual immunotherapy (SLIT) is becoming a preferred form of allergen specific immunotherapy. The efficacy of SLIT is still debated. The purpose of this act of practice trial is to evaluate quality of life outcomes in patients treated with SLIT. Fifty one patients with allergic rhinoconjunctivitis demonstrated by skin testing completed the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) at initiation, at four months and at 10–12 months of SLIT. Significant improvement (P < 0.05) on six of seven domain categories of the RQLQ questionnaire was noted. Total RQLQ scores also showed significant improvement. This study supports SLIT as a modality effective in controlling allergic symptoms

Cognitive function in people with and without freezing of gait in Parkinson’s disease

Freezing of gait (FOG) is common in people with Parkinson’s disease (PD) which is extremely debilitating. One hypothesis for the cause of FOG episodes is impaired cognitive control, however, this is still in debate in the literature. We aimed to assess a comprehensive range of cognitive tests in older adults and people with Parkinson’s with and without FOG and associate FOG severity with cognitive performance. A total of 227 participants took part in the study which included 80 healthy older adults, 81 people with PD who did not have FOG and 66 people with PD and FOG. A comprehensive battery of neuropsychological assessments tested cognitive domains of global cognition, executive function/attention, working memory, and visuospatial function. The severity of FOG was assessed using the new FOG questionnaire and an objective FOG severity score. Cognitive performance was compared between groups using an ANCOVA adjusting for age, gender, years of education and disease severity. Correlations between cognitive performance and FOG severity were analyzed using partial correlations. Cognitive differences were observed between older adults and PD for domains of global cognition, executive function/attention, and working memory. Between those with and without FOG, there were differences for global cognition and executive function/attention, but these differences disappeared when adjusting for covariates. There were no associations between FOG severity and cognitive performance. This study identified no significant difference in cognition between those with and without FOG when adjusting for covariates, particularly disease severity. This may demonstrate that complex rehabilitation programs may be undertaken in those with FOG

Redox chemistry of Perrhenate and other anions

This thesis discusses reduction and oxidation reactions catalysed by the perrhenate anion and oxidation reactions catalysed by other oxo-anions i.e. sulfate and nitrate. Chapter one introduces catalytic hydrosilylation, hydroboration, deoxydehydration and alkene epoxidation and some of their applications, with a focus on high oxidation state complexes. Chapter two describes the synthesis of a salt of perrhenate [N(hexyl)4][ReO4] which is highly soluble in organic solvents. The use of this salt as a catalyst for both the hydrosilylation and hydroboration of carbonyl compounds and carbon dioxide is discussed. Catalytic methylation of amines and anilines with carbon dioxide and hydrosilanes is also reported. Labelled carbon dioxide reactions and DFT calculations are conducted in order to understand the mechanism of carbon dioxide reduction using hydrosilanes. Chapter three outlines the synthesis of a number of alkylammonium and pyridinium perrhenate salts and their application in the deoxydehydration reaction, converting vicinal diols to alkenes. The role of the counterion is discussed with pyridinium perrhenates shown to be more effective catalysts. DFT calculations are conducted to identify the most likely pathway of the catalytic cycle. Alternative reducing agents to the triphenylphosphine initially used are also studied. Chapter four reports results of organic salts of perrhenate, sulfate and nitrate as oxidation catalysts, specifically their ability to catalyse epoxidations of alkenes. By the formation of supramolecular ion pairs (SIPs), these anions are made organic soluble which is found to significantly enhance their catalytic ability, however, the organic counterions used to form these SIPs were found to be of importance. Ionic liquids are also used for the epoxidation of alkenes. Solution studies are presented to further understanding of how these compounds interact with one another in solution. Chapter five contains experimental conditions and characterisation for compounds discussed in this work

Diffusion measurements to understand dynamics and structuring in solutions involving a homologous series of ionic liquids

The self-diffusion coefficients of each of the components in mixtures containing pyridine and each of the homologous series 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imides in acetonitrile were determined using NMR diffusometry (i. e., Pulsed Gradient Spin Echo). The nature of solvation was found to change significantly with the proportion of salt in the mixtures. Increased diffusion coefficients (when corrected for viscosity) for the molecular components were observed with increasing proportion of ionic liquid and with increasing alkyl chain length on the cation. Comparison of the molecular solvents suggests increased interactions in solution of the pyridine with other components of the mixture, consistent with the proposed interactions shown previously to drive changes in reaction kinetics. Discontinuities were seen in the diffusion data for each species in solution across different ionic liquids between the hexyl and octyl derivatives, suggesting a change in the structuring in solution as the alkyl chain on the cation changes and demonstrating the importance of such when considering homologous series

Gene-SGAN: Discovering disease subtypes with imaging and genetic signatures via multi-view weakly-supervised deep clustering

Disease heterogeneity has been a critical challenge for precision diagnosis and treatment, especially in neurologic and neuropsychiatric diseases. Many diseases can display multiple distinct brain phenotypes across individuals, potentially reflecting disease subtypes that can be captured using MRI and machine learning methods. However, biological interpretability and treatment relevance are limited if the derived subtypes are not associated with genetic drivers or susceptibility factors. Herein, we describe Gene-SGAN - a multi-view, weakly-supervised deep clustering method - which dissects disease heterogeneity by jointly considering phenotypic and genetic data, thereby conferring genetic correlations to the disease subtypes and associated endophenotypic signatures. We first validate the generalizability, interpretability, and robustness of Gene-SGAN in semi-synthetic experiments. We then demonstrate its application to real multi-site datasets from 28,858 individuals, deriving subtypes of Alzheimer\u27s disease and brain endophenotypes associated with hypertension, from MRI and single nucleotide polymorphism data. Derived brain phenotypes displayed significant differences in neuroanatomical patterns, genetic determinants, biological and clinical biomarkers, indicating potentially distinct underlying neuropathologic processes, genetic drivers, and susceptibility factors. Overall, Gene-SGAN is broadly applicable to disease subtyping and endophenotype discovery, and is herein tested on disease-related, genetically-associated neuroimaging phenotypes

Extraterrestrial Moessbauer Spectroscopy: More than Three Years of Mars Exploration and Developments for Future Missions

The NASA Mars Exploration Rovers (MER), Spirit and Opportunity, landed on the Red Planet in January 2004. Both rovers are equipped with a miniaturized Moessbauer spectrometer MIMOS II. Designed for a three months mission, both rovers and both Moessbauer instruments are still working after more than three years of exploring the Martian surface. At the beginning of the mission, with a landed intensity of the Moessbauer source of 150 mCi, a 30 minute touch and go measurement produced scientifically valuable data while a good quality Moessbauer spectrum was obtained after approximately eight hours. Now, after about five halflives of the sources have passed, Moessbauer integrations are routinely planned to last approx.48 hours. Because of this and other age-related hardware degradations of the two rover systems, measurements now occur less frequently, but are still of outstanding quality and scientific importance. Summarizing important Moessbauer results, Spirit has traversed the plains from her landing site in Gusev crater and is now, for the greater part of the mission, investigating the stratigraphically older Columbia Hills. Olivine in rocks and soils in the plains suggests that physical rather than chemical processes are currently active

In situ solid-state NMR and XRD studies of the ADOR process and the unusual structure of zeolite IPC-6

R.E.M. and M.N. thank the Royal Society and the E.P.S.R.C. (Grants EP/L014475/1, EP/K025112/1 and EP/K005499/1) for funding work in this area. R.E.M. and J.Č. acknowledge the Czech Science Foundation for the project P106/12/G015 and OP VVV "Excellent Research Teams", project No. CZ.02.1.01/0.0/0.0/15_003/0000417 - CUCAM. S.E.A. would like to thank the ERC (EU FP7 Consolidator Grant 614290 “EXONMR”) and the Royal Society and Wolfson Foundation for a merit award. The UK 850 MHz solid-state NMR Facility used in this research was funded by EPSRC and BBSRC (contract reference PR140003), as well as the University of Warwick including via part funding through Birmingham Science City Advanced Materials Projects 1 and 2 supported by Advantage West Midlands (AWM) and the European Regional Development Fund (ERDF). W.A.S. and D.S.W. acknowledge the Research Council of Norway and NOTUR are acknowledged for providing the computer time at the Norwegian supercomputer facilities (under the project number NN2875k).The assembly–disassembly–organization–reassembly (ADOR) mechanism is a recent method for preparing inorganic framework materials and, in particular, zeolites. This flexible approach has enabled the synthesis of isoreticular families of zeolites with unprecedented continuous control over porosity, and the design and preparation of materials that would have been difficult—or even impossible—to obtain using traditional hydrothermal techniques. Applying the ADOR process to a parent zeolite with the UTL framework topology, for example, has led to six previously unknown zeolites (named IPC-n, where n = 2, 4, 6, 7, 9 and 10). To realize the full potential of the ADOR method, however, a further understanding of the complex mechanism at play is needed. Here, we probe the disassembly, organization and reassembly steps of the ADOR process through a combination of in situ solid-state NMR spectroscopy and powder X-ray diffraction experiments. We further use the insight gained to explain the formation of the unusual structure of zeolite IPC-6.PostprintPeer reviewe

Genomic loci influence patterns of structural covariance in the human brain

Normal and pathologic neurobiological processes influence brain morphology in coordinated ways that give rise to patterns of structural covariance (PSC) across brain regions and individuals during brain aging and diseases. The genetic underpinnings of these patterns remain largely unknown. We apply a stochastic multivariate factorization method to a diverse population of 50,699 individuals (12 studies and 130 sites) and derive data-driven, multi-scale PSCs of regional brain size. PSCs were significantly correlated with 915 genomic loci in the discovery set, 617 of which are newly identified, and 72% were independently replicated. Key pathways influencing PSCs involve reelin signaling, apoptosis, neurogenesis, and appendage development, while pathways of breast cancer indicate potential interplays between brain metastasis and PSCs associated with neurodegeneration and dementia. Using support vector machines, multi-scale PSCs effectively derive imaging signatures of several brain diseases. Our results elucidate genetic and biological underpinnings that influence structural covariance patterns in the human brain