34 research outputs found

    Comparison of different gene-therapy methods to treat Leber hereditary optic neuropathy in a mouse model

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    IntroductionTherapies for Leber hereditary optic neuropathy (LHON), in common with all disorders caused by mutated mitochondrial DNA, are inadequate. We have developed two gene therapy strategies for the disease: mitochondrial-targeted and allotopic expressed and compared them in a mouse model of LHON.MethodsA LHON mouse model was generated by intravitreal injection of a mitochondrialtargeted Adeno-associated virus (AAV) carrying mutant human NADH dehydrogenase 4 gene (hND4/m.11778G>A) to induce retinal ganglion cell (RGC) degeneration and axon loss, the hallmark of the human disease. We then attempted to rescue those mice using a second intravitreal injection of either mitochondrial-targeted or allotopic expressed wildtype human ND4. The rescue of RGCs and their axons were assessed using serial pattern electroretinogram (PERG) and transmission electron microscopy.ResultsCompared to non-rescued LHON controls where PERG amplitude was much reduced, both strategies significantly preserved PERG amplitude over 15 months. However, the rescue effect was more marked with mitochondrial-targeted therapy than with allotopic therapy (p = 0.0128). Post-mortem analysis showed that mitochondrial-targeted human ND4 better preserved small axons that are preferentially lost in human LHON.ConclusionsThese results in a pre-clinical mouse model of LHON suggest that mitochondrially-targeted AAV gene therapy, compared to allotopic AAV gene therapy, is more efficient in rescuing the LHON phenotype

    Effects of a balanced translocation between chromosomes 1 and 11 disrupting the DISC1 locus on white matter integrity

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    Objective Individuals carrying rare, but biologically informative genetic variants provide a unique opportunity to model major mental illness and inform understanding of disease mechanisms. The rarity of such variations means that their study involves small group numbers, however they are amongst the strongest known genetic risk factors for major mental illness and are likely to have large neural effects. DISC1 (Disrupted in Schizophrenia 1) is a gene containing one such risk variant, identified in a single Scottish family through its disruption by a balanced translocation of chromosomes 1 and 11; t(1;11) (q42.1;q14.3). Method Within the original pedigree, we examined the effects of the t(1;11) translocation on white matter integrity, measured by fractional anisotropy (FA). This included family members with (n = 7) and without (n = 13) the translocation, along with a clinical control sample of patients with psychosis (n = 34), and a group of healthy controls (n = 33). Results We report decreased white matter integrity in five clusters in the genu of the corpus callosum, the right inferior fronto-occipital fasciculus, acoustic radiation and fornix. Analysis of the mixed psychosis group also demonstrated decreased white matter integrity in the above regions. FA values within the corpus callosum correlated significantly with positive psychotic symptom severity. Conclusions We demonstrate that the t(1;11) translocation is associated with reduced white matter integrity in frontal commissural and association fibre tracts. These findings overlap with those shown in affected patients with psychosis and in DISC1 animal models and highlight the value of rare but biologically informative mutations in modeling psychosis

    Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans

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    Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686ÔÇô690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r(2) > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 ╬╝g of available chlorine ml(Ôłĺ1) and 500 ╬╝g ml(Ôłĺ1), respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds

    Mixed Effects of Habitat Degradation and Resources on Hantaviruses in Sympatric Wild Rodent Reservoirs within a Neotropical Forest

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    Understanding the ecology of rodent-borne hantaviruses is critical to assessing the risk of spillover to humans. Longitudinal surveys have suggested that hantaviral prevalence in a given host population is tightly linked to rodent ecology and correlates with changes in the species composition of a rodent community over time and/or habitat composition. We tested two hypotheses to identify whether resource addition and/or habitat composition may affect hantavirus prevalence among two sympatric reservoir hosts in a neotropical forest: (i) increased food resources will alter the rodent community and thus hantaviral prevalence; and (ii) host abundance and viral seroprevalence will be associated with habitat composition. We established a baseline of rodent&ndash;virus prevalence in three grid pairs of distinct habitat compositions and subjected one grid of each pair to resource augmentation. Increased rodent species diversity was observed on grids where food was added versus untreated control grids during the first post-treatment sampling session. Resource augmentation changed species community composition, yet it did not affect the prevalence of hantavirus in the host population over time, nor was there evidence of a dilution effect. Secondly, we show that the prevalence of the virus in the respective reservoir hosts was associated with habitat composition at two spatial levels, independent of resource addition, supporting previous findings that habitat composition is a primary driver of the prevalence of hantaviruses in the neotropics

    Plasticizers increase adhesion of the deteriogenic fungus Aureobasidium pullulans to polyvinyl chloride

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    Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10(8) blastospores ml(-1). That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13 degrees in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of A. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces

    Habitat, species richness and hantaviruses of sigmodontine rodents within the Interior Atlantic Forest, Paraguay

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    <div><p>Four of the nine sigmodontine tribes have species that serve as reservoirs of rodent-borne hantaviruses (RBO-HV), few have been studied in any depth. Several viruses have been associated with human cases of hantavirus pulmonary syndrome often through peridomestic exposure. Jabora (JABV) and Juquitiba (JUQV), harbored by <i>Akodon montensis</i> and <i>Oligoryzomys nigripes</i>, respectively, are endemic and sympatric in the Reserva Natural de Bosque Mbaracay├║ (RNBM), Paraguay, a protected area of the Interior Atlantic Forest. Rodent communities were surveyed along a 30 km stretch of the RNBM in eight vegetation classifications (Low, High, Bamboo, Riparian and Liana Forests, Bamboo Understory, Cerrado, and Meadow/Grasslands). We collected 417 rodents from which 11 species were identified; <i>Akodon montensis</i> was the predominant species (72%; 95%CI: 64.7%-76.3%), followed by <i>Hylaeamys megacephalus</i> (15% (11.2%-18.2%)) and <i>Oligoryzomys nigripes</i> (9% (6.6%-12.4%)). We examined the statistical associations among habitat (vegetation class) type, rodent species diversity, population structure (age, sex, and weight), and prevalence of RBO-HV antibody and/or viral RNA (Ab/RNA) or characteristic <i>Leishmania</i> tail lesions. Ab/RNA positive rodents were not observed in Cerrado and Low Forest. <i>A</i>. <i>montensis</i> had an overall Ab/RNA prevalence of 7.7% (4.9%-11.3%) and <i>O</i>. <i>nigripes</i> had an overall prevalence of 8.6% (1.8%-23.1%). For <i>A</i>. <i>montensis</i>, the odds of being Ab/RNA positive in High Forest was 3.73 times of the other habitats combined. There was no significant difference among age classes in the proportion of Ab/RNA positive rodents overall (p = 0.66), however, all 11 RNA-positive individuals were adult. Sex and habitat had independent prognostic value for hantaviral Ab/RNA in the study population; age, presence of tail scar/lesion (19% of the rodents) and weight did not. Adjusting for habitat, female rodents had less risk of becoming infected. Importantly, these data suggest habitat preferences of two sympatric rodent reservoirs for two endemic hantaviruses and the importance of including habitat in models of species diversity and habitat fragmentation.</p></div