2,115 research outputs found

    Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824.

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    Background: Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome. Results: We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis. Conclusions: We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes. © 2013 Kovács et al.; licensee BioMed Central Ltd

    The performance of plant species in removing nutrients from stormwater in biofiltration systems in Cape Town

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    In 2009, the City of Cape Town (CoCT) adopted a stormwater policy which mandates that new and existing developments should reduce the concentration of phosphorus and suspended solids in stormwater runoff by 45% and 80%, respectively, but offered no explicit guidance about how these water quality targets might be achieved. This study aims to contribute to the limited knowledge that exists about the performance of local plant species to treat stormwater. A large nursery-based study was conducted to investigate the performance of 9 locally-occurring plant species to remove orthophosphate (PO4-3), ammonia (NH3) and nitrate (NO3-) found in urban stormwater. Synthetic stormwater was applied to each species as well as a control consisting only of soil (Malmesbury shale). The discharge was collected from a drainage pipe at the base of each of the 150 containers. The results show that all species (excluding Ficinia) reduced the average concentrations of PO4-3 by 81% and NH3 by 90%. By contrast, NO3- was reduced by an average of 69% (excluding by Elegia and Phragmites) with 8 of the 9 species removing significantly more than the control. The species that performed well for all three nutrients include Agapanthus and turf grasses, Stenotaphrum and Pennisetum. The results of the study highlight three important factors in the design of biofilters: that a substantial proportion of nutrients can be captured or absorbed by plants; that the soil medium is an important factor in the removal of PO4-3 and NH3; and that plant choice is essential in the removal of NO3-. Future research should test plant species in both the laboratory and field settings, and should include additional contaminants such as household detergents, heavy metals and bacteria

    Investigating white matter fibre density and morphology using fixel-based analysis

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    Voxel-based analysis of diffusion MRI data is increasingly popular. However, most white matter voxels contain contributions from multiple fibre populations (often referred to as crossing fibres), and therefore voxel-averaged quantitative measures (e.g. fractional anisotropy) are not fibre-specific and have poor interpretability. Using higher-order diffusion models, parameters related to fibre density can be extracted for individual fibre populations within each voxel (‘fixels’), and recent advances in statistics enable the multi-subject analysis of such data. However, investigating within-voxel microscopic fibre density alone does not account for macroscopic differences in the white matter morphology (e.g. the calibre of a fibre bundle). In this work, we introduce a novel method to investigate the latter, which we call fixel-based morphometry (FBM). To obtain a more complete measure related to the total number of white matter axons, information from both within-voxel microscopic fibre density and macroscopic morphology must be combined. We therefore present the FBM method as an integral piece within a comprehensive fixel-based analysis framework to investigate measures of fibre density, fibre-bundle morphology (cross-section), and a combined measure of fibre density and cross-section. We performed simulations to demonstrate the proposed measures using various transformations of a numerical fibre bundle phantom. Finally, we provide an example of such an analysis by comparing a clinical patient group to a healthy control group, which demonstrates that all three measures provide distinct and complementary information. By capturing information from both sources, the combined fibre density and cross-section measure is likely to be more sensitive to certain pathologies and more directly interpretable

    Quantifying single nucleotide variant detection sensitivity in exome sequencing

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    BACKGROUND: The targeted capture and sequencing of genomic regions has rapidly demonstrated its utility in genetic studies. Inherent in this technology is considerable heterogeneity of target coverage and this is expected to systematically impact our sensitivity to detect genuine polymorphisms. To fully interpret the polymorphisms identified in a genetic study it is often essential to both detect polymorphisms and to understand where and with what probability real polymorphisms may have been missed. RESULTS: Using down-sampling of 30 deeply sequenced exomes and a set of gold-standard single nucleotide variant (SNV) genotype calls for each sample, we developed an empirical model relating the read depth at a polymorphic site to the probability of calling the correct genotype at that site. We find that measured sensitivity in SNV detection is substantially worse than that predicted from the naive expectation of sampling from a binomial. This calibrated model allows us to produce single nucleotide resolution SNV sensitivity estimates which can be merged to give summary sensitivity measures for any arbitrary partition of the target sequences (nucleotide, exon, gene, pathway, exome). These metrics are directly comparable between platforms and can be combined between samples to give “power estimates” for an entire study. We estimate a local read depth of 13X is required to detect the alleles and genotype of a heterozygous SNV 95% of the time, but only 3X for a homozygous SNV. At a mean on-target read depth of 20X, commonly used for rare disease exome sequencing studies, we predict 5–15% of heterozygous and 1–4% of homozygous SNVs in the targeted regions will be missed. CONCLUSIONS: Non-reference alleles in the heterozygote state have a high chance of being missed when commonly applied read coverage thresholds are used despite the widely held assumption that there is good polymorphism detection at these coverage levels. Such alleles are likely to be of functional importance in population based studies of rare diseases, somatic mutations in cancer and explaining the “missing heritability” of quantitative traits

    Cryotomography of budding influenza a virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end

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    Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process

    The critical role of ERK in death resistance and invasiveness of hypoxia-selected glioblastoma cells

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    <p>Abstract</p> <p>Background</p> <p>The rapid growth of tumor parenchyma leads to chronic hypoxia that can result in the selection of cancer cells with a more aggressive behavior and death-resistant potential to survive and proliferate. Thus, identifying the key molecules and molecular mechanisms responsible for the phenotypic changes associated with chronic hypoxia has valuable implications for the development of a therapeutic modality. The aim of this study was to identify the molecular basis of the phenotypic changes triggered by chronic repeated hypoxia.</p> <p>Methods</p> <p>Hypoxia-resistant T98G (HRT98G) cells were selected by repeated exposure to hypoxia and reoxygenation. Cell death rate was determined by the trypan blue exclusion method and protein expression levels were examined by western blot analysis. The invasive phenotype of the tumor cells was determined by the Matrigel invasion assay. Immunohistochemistry was performed to analyze the expression of proteins in the brain tumor samples. The Student T-test and Pearson Chi-Square test was used for statistical analyses.</p> <p>Results</p> <p>We demonstrate that chronic repeated hypoxic exposures cause T98G cells to survive low oxygen tension. As compared with parent cells, hypoxia-selected T98G cells not only express higher levels of anti-apoptotic proteins such as Bcl-2, Bcl-X<sub>L</sub>, and phosphorylated ERK, but they also have a more invasive potential in Matrigel invasion chambers. Activation or suppression of ERK pathways with a specific activator or inhibitor, respectively, indicates that ERK is a key molecule responsible for death resistance under hypoxic conditions and a more invasive phenotype. Finally, we show that the activation of ERK is more prominent in malignant glioblastomas exposed to hypoxia than in low grade astrocytic glial tumors.</p> <p>Conclusion</p> <p>Our study suggests that activation of ERK plays a pivotal role in death resistance under chronic hypoxia and phenotypic changes related to the invasive phenotype of HRT98G cells compared to parent cells.</p

    Network model of immune responses reveals key effectors to single and co-infection dynamics by a respiratory bacterium and a gastrointestinal helminth

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    Co-infections alter the host immune response but how the systemic and local processes at the site of infection interact is still unclear. The majority of studies on co-infections concentrate on one of the infecting species, an immune function or group of cells and often focus on the initial phase of the infection. Here, we used a combination of experiments and mathematical modelling to investigate the network of immune responses against single and co-infections with the respiratory bacterium Bordetella bronchiseptica and the gastrointestinal helminth Trichostrongylus retortaeformis. Our goal was to identify representative mediators and functions that could capture the essence of the host immune response as a whole, and to assess how their relative contribution dynamically changed over time and between single and co-infected individuals. Network-based discrete dynamic models of single infections were built using current knowledge of bacterial and helminth immunology; the two single infection models were combined into a co-infection model that was then verified by our empirical findings. Simulations showed that a T helper cell mediated antibody and neutrophil response led to phagocytosis and clearance of B. bronchiseptica from the lungs. This was consistent in single and co-infection with no significant delay induced by the helminth. In contrast, T. retortaeformis intensity decreased faster when co-infected with the bacterium. Simulations suggested that the robust recruitment of neutrophils in the co-infection, added to the activation of IgG and eosinophil driven reduction of larvae, which also played an important role in single infection, contributed to this fast clearance. Perturbation analysis of the models, through the knockout of individual nodes (immune cells), identified the cells critical to parasite persistence and clearance both in single and co-infections. Our integrated approach captured the within-host immuno-dynamics of bacteria-helminth infection and identified key components that can be crucial for explaining individual variability between single and co-infections in natural populations

    Risk-taking attitudes and their association with process and outcomes of cardiac care: a cohort study

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    <p>Abstract</p> <p>Background</p> <p>Prior research reveals that processes and outcomes of cardiac care differ across sociodemographic strata. One potential contributing factor to such differences is the personality traits of individuals within these strata. We examined the association between risk-taking attitudes and cardiac patients' clinical and demographic characteristics, the likelihood of undergoing invasive cardiac procedures and survival.</p> <p>Methods</p> <p>We studied a large inception cohort of patients who underwent cardiac catheterization between July 1998 and December 2001. Detailed clinical and demographic data were collected at time of cardiac catheterization and through a mailed survey one year post-catheterization. The survey included three general risk attitude items from the Jackson Personality Inventory. Patients' (n = 6294) attitudes toward risk were categorized as risk-prone versus non-risk-prone and were assessed for associations with baseline clinical and demographic characteristics, treatment received (i.e., medical therapy, coronary artery bypass graft (CABG) surgery, percutaneous coronary intervention (PCI)), and survival (to December 2005).</p> <p>Results</p> <p>2827 patients (45%) were categorized as risk-prone. Having risk-prone attitudes was associated with younger age (p < .001), male sex (p < .001), current smoking (p < .001) and higher household income (p < .001). Risk-prone patients were more likely to have CABG surgery in unadjusted (Odds Ratio [OR] = 1.21; 95% CI 1.08–1.36) and adjusted (OR = 1.18; 95% CI 1.02–1.36) models, but were no more likely to have PCI or any revascularization. Having risk-prone attitudes was associated with better survival in an unadjusted survival analysis (Hazard Ratio [HR] = 0.78 (95% CI 0.66–0.93), but not in a risk-adjusted analysis (HR = 0.92, 95% CI 0.77–1.10).</p> <p>Conclusion</p> <p>These exploratory findings suggest that patient attitudes toward risk taking may <b>contribute to </b>some of the documented differences in use of invasive cardiac procedures. An awareness of these associations could help healthcare providers as they counsel patients regarding cardiac care decisions.</p
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