Dynamic Modelling under Uncertainty: The Case of Trypanosoma brucei Energy Metabolism

Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis). Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies

Kinetic modeling of tricarboxylic acid cycle and glyoxylate bypass in Mycobacterium tuberculosis, and its application to assessment of drug targets

BACKGROUND: Targeting persistent tubercule bacilli has become an important challenge in the development of anti-tuberculous drugs. As the glyoxylate bypass is essential for persistent bacilli, interference with it holds the potential for designing new antibacterial drugs. We have developed kinetic models of the tricarboxylic acid cycle and glyoxylate bypass in Escherichia coli and Mycobacterium tuberculosis, and studied the effects of inhibition of various enzymes in the M. tuberculosis model. RESULTS: We used E. coli to validate the pathway-modeling protocol and showed that changes in metabolic flux can be estimated from gene expression data. The M. tuberculosis model reproduced the observation that deletion of one of the two isocitrate lyase genes has little effect on bacterial growth in macrophages, but deletion of both genes leads to the elimination of the bacilli from the lungs. It also substantiated the inhibition of isocitrate lyases by 3-nitropropionate. On the basis of our simulation studies, we propose that: (i) fractional inactivation of both isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 is required for a flux through the glyoxylate bypass in persistent mycobacteria; and (ii) increasing the amount of active isocitrate dehydrogenases can stop the flux through the glyoxylate bypass, so the kinase that inactivates isocitrate dehydrogenase 1 and/or the proposed inactivator of isocitrate dehydrogenase 2 is a potential target for drugs against persistent mycobacteria. In addition, competitive inhibition of isocitrate lyases along with a reduction in the inactivation of isocitrate dehydrogenases appears to be a feasible strategy for targeting persistent mycobacteria. CONCLUSION: We used kinetic modeling of biochemical pathways to assess various potential anti-tuberculous drug targets that interfere with the glyoxylate bypass flux, and indicated the type of inhibition needed to eliminate the pathogen. The advantage of such an approach to the assessment of drug targets is that it facilitates the study of systemic effect(s) of the modulation of the target enzyme(s) in the cellular environment

Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE Collaboration): a meta-analysis of genome-wide association studies

<p>Background - Various genome-wide association studies (GWAS) have been done in ischaemic stroke, identifying a few loci associated with the disease, but sample sizes have been 3500 cases or less. We established the METASTROKE collaboration with the aim of validating associations from previous GWAS and identifying novel genetic associations through meta-analysis of GWAS datasets for ischaemic stroke and its subtypes.</p> <p>Methods - We meta-analysed data from 15 ischaemic stroke cohorts with a total of 12 389 individuals with ischaemic stroke and 62 004 controls, all of European ancestry. For the associations reaching genome-wide significance in METASTROKE, we did a further analysis, conditioning on the lead single nucleotide polymorphism in every associated region. Replication of novel suggestive signals was done in 13 347 cases and 29 083 controls.</p> <p>Findings - We verified previous associations for cardioembolic stroke near PITX2 (p=2·8×10−16) and ZFHX3 (p=2·28×10−8), and for large-vessel stroke at a 9p21 locus (p=3·32×10−5) and HDAC9 (p=2·03×10−12). Additionally, we verified that all associations were subtype specific. Conditional analysis in the three regions for which the associations reached genome-wide significance (PITX2, ZFHX3, and HDAC9) indicated that all the signal in each region could be attributed to one risk haplotype. We also identified 12 potentially novel loci at p<5×10−6. However, we were unable to replicate any of these novel associations in the replication cohort.</p> <p>Interpretation - Our results show that, although genetic variants can be detected in patients with ischaemic stroke when compared with controls, all associations we were able to confirm are specific to a stroke subtype. This finding has two implications. First, to maximise success of genetic studies in ischaemic stroke, detailed stroke subtyping is required. Second, different genetic pathophysiological mechanisms seem to be associated with different stroke subtypes.</p&gt

LC–MS-based absolute metabolite quantification:Application to metabolic flux measurement in trypanosomes

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism

Reduced haemodynamic response in the ageing visual cortex measured by absolute fNIRS

The effect of healthy ageing on visual cortical activation is still to be fully explored. This study aimed to elucidate whether the haemodynamic response (HDR) of the visual cortex altered as a result of ageing. Visually normal (healthy) participants were presented with a simple visual stimulus (reversing checkerboard). Full optometric screening was implemented to identify two age groups: younger adults (n = 12, mean age 21) and older adults (n = 13, mean age 71). Frequency-domain Multi-distance (FD-MD) functional Near-Infrared Spectroscopy (fNIRS) was used to measure absolute changes in oxygenated [HbO] and deoxygenated [HbR] haemoglobin concentrations in the occipital cortices. Utilising a slow event-related design, subjects viewed a full field reversing checkerboard with contrast and check size manipulations (15 and 30 minutes of arc, 50% and 100% contrast). Both groups showed the characteristic response of increased [HbO] and decreased [HbR] during stimulus presentation. However, older adults produced a more varied HDR and often had comparable levels of [HbO] and [HbR] during both stimulus presentation and baseline resting state. Younger adults had significantly greater concentrations of both [HbO] and [HbR] in every investigation regardless of the type of stimulus displayed (p<0.05). The average variance associated with this age-related effect for [HbO] was 88% and [HbR] 91%. Passive viewing of a visual stimulus, without any cognitive input, showed a marked age-related decline in the cortical HDR. Moreover, regardless of stimulus parameters such as check size, the HDR was characterised by age. In concurrence with present neuroimaging literature, we conclude that the visual HDR decreases as healthy ageing proceeds

Immunization with one Theileria parva strain results in similar level of CTL strain-specificity and protection compared to immunization with the three-component Muguga cocktail in MHC-matched animals

Abstract Background The tick-borne protozoan parasite Theileria parva causes a usually fatal cattle disease known as East Coast fever in sub-Saharan Africa, with devastating consequences for poor small-holder farmers. Immunity to T. parva, believed to be mediated by a cytotoxic T lymphocyte (CTL) response, is induced following natural infection and after vaccination with a live vaccine, known as the Infection and Treatment Method (ITM). The most commonly used version of ITM is a combination of parasites derived from three isolates (Muguga, Kiambu 5 and Serengeti-transformed), known as the “Muguga cocktail”. The use of a vaccine comprising several strains is believed to be required to induce a broad immune response effective against field challenge. In this study we investigated whether immunization with the Muguga cocktail induces a broader CTL response than immunization with a single strain (Muguga). Results Four MHC haplotype-matched pairs of cattle were immunized with either the trivalent Muguga cocktail or the single Muguga strain. CTL specificity was assessed on a panel of five different strains, and clonal responses to these strains were also assessed in one of the MHC-matched pairs. We did not find evidence for a broader CTL response in animals immunized with the Muguga cocktail compared to those immunized with the Muguga strain alone, in either the bulk or clonal CTL analyses. This was supported by an in vivo trial in which all vaccinated animals survived challenge with a lethal dose of the Muguga cocktail vaccine stabilate. Conclusion We did not observe any substantial differences in the immunity generated from animals immunized with either Muguga alone or the Muguga cocktail in the animals tested here, corroborating earlier results showing limited antigenic diversity in the Muguga cocktail. These results may warrant further field studies using single T. parva strains as future vaccine candidates

Consistent phenological shifts in the making of a biodiversity hotspot: the Cape flora

Background The best documented survival responses of organisms to past climate change on short (glacial-interglacial) timescales are distributional shifts. Despite ample evidence on such timescales for local adaptations of populations at specific sites, the long-term impacts of such changes on evolutionary significant units in response to past climatic change have been little documented. Here we use phylogenies to reconstruct changes in distribution and flowering ecology of the Cape flora - South Africa's biodiversity hotspot - through a period of past (Neogene and Quaternary) changes in the seasonality of rainfall over a timescale of several million years. Results Forty-three distributional and phenological shifts consistent with past climatic change occur across the flora, and a comparable number of clades underwent adaptive changes in their flowering phenology (9 clades; half of the clades investigated) as underwent distributional shifts (12 clades; two thirds of the clades investigated). Of extant Cape angiosperm species, 14-41% have been contributed by lineages that show distributional shifts consistent with past climate change, yet a similar proportion (14-55%) arose from lineages that shifted flowering phenology. Conclusions Adaptive changes in ecology at the scale we uncover in the Cape and consistent with past climatic change have not been documented for other floras. Shifts in climate tolerance appear to have been more important in this flora than is currently appreciated, and lineages that underwent such shifts went on to contribute a high proportion of the flora's extant species diversity. That shifts in phenology, on an evolutionary timescale and on such a scale, have not yet been detected for other floras is likely a result of the method used; shifts in flowering phenology cannot be detected in the fossil record

Targeting pathogen metabolism without collateral damage to the host

The development of drugs that can inactivate disease-causing cells (e.g. cancer cells or parasites) without causing collateral damage to healthy or to host cells is complicated by the fact that many proteins are very similar between organisms. Nevertheless, due to subtle, quantitative differences between the biochemical reaction networks of target cell and host, a drug can limit the flux of the same essential process in one organism more than in another. We identified precise criteria for this â €network-based' drug selectivity, which can serve as an alternative or additive to structural differences. We combined computational and experimental approaches to compare energy metabolism in the causative agent of sleeping sickness, Trypanosoma brucei, with that of human erythrocytes, and identified glucose transport and glyceraldehyde-3-phosphate dehydrogenase as the most selective antiparasitic targets. Computational predictions were validated experimentally in a novel parasite-erythrocytes co-culture system. Glucose-transport inhibitors killed trypanosomes without killing erythrocytes, neurons or liver cells

Integrated approach to prevent functional decline in hospitalized elderly: the Prevention and Reactivation Care Program (PReCaP)

<p>Abstract</p> <p>Background</p> <p>Hospital related functional decline in older patients is an underestimated problem. Thirty-five procent of 70-year old patients experience functional decline during hospital admission in comparison with pre-illness baseline. This percentage increases considerably with age.</p> <p>Methods/design</p> <p>To address this issue, the Vlietland Ziekenhuis in The Netherlands has implemented an innovative program (PReCaP), aimed at reducing hospital related functional decline among elderly patients by offering interventions that are multidisciplinary, integrated and goal-oriented at the physical, social, and psychological domains of functional decline.</p> <p>Discussion</p> <p>This paper presents a detailed description of the intervention, which incorporates five distinctive elements: (1) Early identification of elderly patients with a high risk of functional decline, and if necessary followed by the start of the reactivation treatment within 48 h after hospital admission; (2) Intensive follow-up treatment for a selected patient group at the Prevention and Reactivation Centre (PRC); (3) Availability of multidisciplinary geriatric expertise; (4) Provision of support and consultation of relevant professionals to informal caregivers; (5) Intensive follow-up throughout the entire chain of care by a casemanager with geriatric expertise. Outcome and process evaluations are ongoing and results will be published in a series of future papers.</p> <p>Trial registration</p> <p>The Netherlands National Trial Register: <a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2317">NTR2317</a></p

Post-transplant lymphoproliferative disorders and Epstein-Barr virus DNAemia in a cohort of lung transplant recipients

<p>Abstract</p> <p>Background</p> <p>Post-transplant lymphoproliferative disorders (PTLD) are serious complications in lung transplant recipients. No consensus on EBV DNAemia levels predictive of PTLD has been reached. In addition, in many instances EBV DNAemia is determined in patients with suggestive symptoms only.</p> <p>Methods</p> <p>The characteristics of five patients with PTLD as well as the prevalence of EBV DNAmia in a cohort of 137 consecutive patients receiving lung transplantation are described.</p> <p>Results</p> <p>Twenty-six out of 137 patients (18.9%) were excluded from the analysis because lost at follow-up or dead from PTLD-independent reasons within three months of transplantation. EBV DNA in peripheral blood mononuclear cells (PBMC) was determined in 83/111 patients (74.8%) because of potential PTLD-related symptoms, while 28 patients (25.2%) showed no symptoms and were not examined. EBV DNAemia was positive in 53/83 patients (63.8%), and negative in 30/83 patients (36.2%). PTLD was diagnosed in five (4.5%) patients at a median time of 270 (range 120-870) days following transplantation. All five PTLD (three large B-cell lymphomas, one Hodgkin lymphoma and one possible pre-neoplastic lesion) were potentially associated with EBV infection. However, only 3/5 patients with PTLD had detectable EBV DNAemia: < 1,000 copies EBV DNA/1 × 10⁵PBMC in one patient and > 1,000 copies EBV DNA/1 × 10⁵PBMC in two patients.</p> <p>Conclusion</p> <p>A systematic multidisciplinary (clinical, radiologic, virologic and histologic) approach is mandatory for the diagnosis and management of PTLD in lung transplant recipients, while monitoring of symptomatic patients only may provide an incomplete or late picture of the clinical problem. In addition, staining for EBV antigens and quantification of EBV DNA in biopsy specimens should always be performed to understand the role of EBV infection in the pathogenesis of PTLD.</p