3,458 research outputs found

    Investigating the pathophysiology of KCNJ13 and USH2A retinopathies using zebrafish models

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    Inherited retinal diseases (IRDs) encompass a large group of clinically and genetically heterogeneous diseases that affect approximately 1 in 3000 people, representing an important cause of severe visual loss in the human population. In the past two decades, significant contributions towards our understanding of these disorders has included the identification of over 250 causative genes. In this thesis, the pathophysiology of IRDs associated with KCNJ13 and USH2A was investigated using relevant zebrafish models. Mutations in KCNJ13 are associated with Leber congenital amaurosis, the most severe IRD subtype that causes blindness in childhood. KCNJ13 encodes the Kir7.1 channel, a potassium channel expressed on the apical retinal pigment epithelium (RPE). Longitudinal assessment of the kcnj13 mutant zebrafish (obelixtd15) revealed a late onset retinal degeneration at 12 months with retinovascular abnormalities, and a corresponding decline of visual function. Ultrastructural examination of the obelixtd15 RPE uncovered changes in phagosome clearance and mitochondrial growth prior to notable degeneration which indicate that the disease may be a primary phagosome failure with a secondary failure in mitochondrial physiology. In addition, gene and protein expression changes consistent with altered mitochondrial activity and retinal stress were observed. The alterations in the RPE are reminiscent of those seen in age-related macular degeneration and highlight potential therapeutic targets for KCNJ13 retinopathy. USH2A mutations are the most common cause of Usher Syndrome, characterised by combined retinitis pigmentosa and sensorineural deafness. USH2A encodes the large transmembrane protein, usherin, expressed in the photoreceptors and cochlear hair cells. CRISPR/Cas9 gene targeting was used to create an ush2a mutant zebrafish line, which showed a slowly progressive photoreceptor degeneration from 6 months with mislocalisation of rhodopsin. Developmental abnormalities in the neuromast and inner ear hair cells were also identified in ush2a larvae. The findings suggest that ush2a is involved in rhodopsin trafficking and rod photoreceptor maintenance in the zebrafish retina, as well as playing a role in mechanosensory system development. Overall, the ush2a zebrafish phenotype showed consistency with the clinical phenotype in USH2A patients, indicating the value of this model for gaining further insight into the disease pathophysiology

    Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3): Role in Retinal Development and Disease

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    NR2E3 is a nuclear hormone receptor gene required for the correct development of the retinal rod photoreceptors. Expression of NR2E3 protein in rod cell precursors suppresses cone-specific gene expression and, in concert with other transcription factors including NRL, activates the expression of rod-specific genes. Pathogenic variants involving NR2E3 cause a spectrum of retinopathies, including enhanced S-cone syndrome, Goldmann–Favre syndrome, retinitis pigmentosa, and clumped pigmentary retinal degeneration, with limited evidence of genotype–phenotype correlations. A common feature of NR2E3-related disease is an abnormally high number of cone photoreceptors that are sensitive to short wavelength light, the S-cones. This characteristic has been supported by mouse studies, which have also revealed that loss of Nr2e3 function causes photoreceptors to develop as cells that are intermediate between rods and cones. While there is currently no available cure for NR2E3-related retinopathies, there are a number of emerging therapeutic strategies under investigation, including the use of viral gene therapy and gene editing, that have shown promise for the future treatment of patients with NR2E3 variants and other inherited retinal diseases. This review provides a detailed overview of the current understanding of the role of NR2E3 in normal development and disease, and the associated clinical phenotypes, animal models, and therapeutic studies

    Comparative ethnoentomology of edible stinkbugs in southern Africa and sustainable management considerations

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    Insects, such as stinkbugs, are able to produce noxious defence chemicals to ward off predators, nevertheless, some ethnic groups have recipes to render them delicious. We provide an example of edible stinkbugs (Encosternum delegorguei) used by two locally separate ethnic groups in South Africa, the Vhavenda and Mapulana, with a third group, the Bolobedu using them for commercial purposes. Structured interview schedules and observations with 106 harvesters were conducted to determine differences in use, nomenclature and oral history, methods of collection and preparation as well as perceptions pertaining to availability. The stinkbugs’ foul defence chemical and flight response necessitates nocturnal harvesting when the insect is immobilised by cold. The defence chemical stains the skin and affects vision yet protective gear is not worn. Damage to host trees was recorded when harvesters poached from plantations or private land, whereas, in communal-lands, sustainable methods were preferred. The legitimisation of stinkbug harvesting and introduction of a collection funnel could reduce conflicts with managers of plantations and private land. Two methods to remove the defence chemical for increased palatability were used. Preparation methods differed in whether or not water was used and also whether the head was left intact or removed. Stinkbugs have numerous medicinal uses, in particular as a hangover cure. Awareness and optimal use of beneficial insects, such as stinkbugs, in rural areas could lead to a reconsideration of current environmental management strategies, where harvesters act as habitat stewards and clearing, grazing or burning indigenous vegetation is kept to a minimum.The National Research Foundation (NRF) of South Africa provided funding in the Indigenous Knowledge Systems Focus Area for the project on Insects in Culture and Medicine (FA2006032900013)

    Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia

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    Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes

    Successful large gene augmentation of USH2A with non-viral episomal vectors

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    USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development

    Changes in Mitochondrial Size and Morphology in the RPE and Photoreceptors of the Developing and Ageing Zebrafish

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    Mitochondria are essential adenosine triphosphate (ATP)-generating cellular organelles. In the retina, they are highly numerous in the photoreceptors and retinal pigment epithelium (RPE) due to their high energetic requirements. Fission and fusion of the mitochondria within these cells allow them to adapt to changing demands over the lifespan of the organism. Using transmission electron microscopy, we examined the mitochondrial ultrastructure of zebrafish photoreceptors and RPE from 5 days post fertilisation (dpf) through to late adulthood (3 years). Notably, mitochondria in the youngest animals were large and irregular shaped with a loose cristae architecture, but by 8 dpf they had reduced in size and expanded in number with more defined cristae. Investigation of temporal gene expression of several mitochondrial-related markers indicated fission as the dominant mechanism contributing to the changes observed over time. This is likely to be due to continued mitochondrial stress resulting from the oxidative environment of the retina and prolonged light exposure. We have characterised retinal mitochondrial ageing in a key vertebrate model organism, that provides a basis for future studies of retinal diseases that are linked to mitochondrial dysfunction

    A Standardised Format for Exchanging User Study Instruments

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    Increasing re-use in Interactive Information Retrieval (IIR) has been an ongoing aim in IIR for a significant amount of time, however progress has been limited and patchy. While re-use of some study aspects can be difficult due to the varied nature of IIR studies, the use of pre- and post-task self-reported measures is widespread and relatively standardised. Nevertheless, re-use of elements in this area is also limited, in part because systems used to implement them are not able to exchange question, instruments, or complete study setups. To address this, this paper presents a standardised, but extendable, format for IIR survey instrument exchange

    Identification of novel coloboma candidate genes through conserved gene expression analyses across four vertebrate species

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    Ocular coloboma (OC) is a failure of complete optic fissure closure during embryonic development and presents as a tissue defect along the proximal–distal axis of the ventral eye. It is classed as part of the clinical spectrum of structural eye malformations with microphthalmia and anophthalmia, collectively abbreviated to MAC. Despite deliberate attempts to identify causative variants in MAC, many patients remain without a genetic diagnosis. To reveal potential candidate genes, we utilised transcriptomes experimentally generated from embryonic eye tissues derived from humans, mice, zebrafish, and chicken at stages coincident with optic fissure closure. Our in-silico analyses found 10 genes with optic fissure-specific enriched expression: ALDH1A3, BMPR1B, EMX2, EPHB3, NID1, NTN1, PAX2, SMOC1, TENM3, and VAX1. In situ hybridization revealed that all 10 genes were broadly expressed ventrally in the developing eye but that only PAX2 and NTN1 were expressed in cells at the edges of the optic fissure margin. Of these conserved optic fissure genes, EMX2, NID1, and EPHB3 have not previously been associated with human MAC cases. Targeted genetic manipulation in zebrafish embryos using CRISPR/Cas9 caused the developmental MAC phenotype for emx2 and ephb3. We analysed available whole genome sequencing datasets from MAC patients and identified a range of variants with plausible causality. In combination, our data suggest that expression of genes involved in ventral eye development is conserved across a range of vertebrate species and that EMX2, NID1, and EPHB3 are candidate loci that warrant further functional analysis in the context of MAC and should be considered for sequencing in cohorts of patients with structural eye malformations

    PHEME : computing veracity : the fourth challenge of big social data

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    The veracity of information spreading through social media can sometimes be hard to establish and the deliberate or accidental spread of false information, especially during natural disasters or emergencies, is quite common. We coined the term phemes to describe fast spreading memes which are enhanced with truthfulness information. The PHEME project (http://www.pheme.eu) attempts to identify in real-time four kinds of phemes: controversy, speculation, misinformation and disinformation. This brings challenges in modelling the social network spread of and the online conversations around phemes; developing rumour detection methods; and using historical data to model trustworthiness of the information source

    Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

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    This paper presents measurements of the W+→Ό+ÎœW^+ \rightarrow \mu^+\nu and W−→Ό−ΜW^- \rightarrow \mu^-\nu cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13
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