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

Mapping the metabolism of five amino acids in bloodstream form Trypanosoma brucei using U- 13C-labelled substrates and LC–MS

The metabolism of the parasite Trypanosoma brucei has been the focus of numerous studies since the 1940s. Recently it was shown, using metabolomics coupled with heavy-atom isotope labelled glucose, that the metabolism of the bloodstream form parasite is more complex than previously thought. The present study also raised a number of questions regarding the origin of several metabolites, for example succinate, only a proportion of which derives from glucose. In order to answer some of these questions and explore the metabolism of bloodstream form T. brucei in more depth we followed the fate of five heavy labelled amino acids – glutamine, proline, methionine, cysteine and arginine – using an LC–MS based metabolomics approach. We found that some of these amino acids have roles beyond those previously thought and we have tentatively identified some unexpected metabolites which need to be confirmed and their function determined

Bayesian Estimation Of Performance Measures Of Cervical Cancer Screening Tests In The Presence Of Covariates And Absence Of A Gold Standard

In this paper we develop a Bayesian analysis to estimate the disease prevalence, the sensitivity and specificity of three cervical cancer screening tests (cervical cytology, visual inspection with acetic acid and Hybrid Capture II) in the presence of a covariate and in the absence of a gold standard. We use Metropolis-Hastings algorithm to obtain the posterior summaries of interest. The estimated prevalence of cervical lesions was 6.4% (a 95% credible interval [95% CI] was 3.9, 9.3). The sensitivity of cervical cytology (with a result of ≥ ASC-US) was 53.6% (95% CI: 42.1, 65.0) compared with 52.9% (95% CI: 43.5, 62.5) for visual inspection with acetic acid and 90.3% (95% CI: 76.2, 98.7) for Hybrid Capture II (with result of >1 relative light units). The specificity of cervical cytology was 97.0% (95% CI: 95.5, 98.4) and the specifi cities for visual inspection with acetic acid and Hybrid Capture II were 93.0% (95% CI: 91.0, 94.7) and 88.7% (95% CI: 85.9, 91.4), respectively. The Bayesian model with covariates suggests that the sensitivity and the specificity of the visual inspection with acetic acid tend to increase as the age of the women increases. The Bayesian method proposed here is an useful alternative to estimate measures of performance of diagnostic tests in the presence of covariates and when a gold standard is not available. An advantage of the method is the fact that the number of parameters to be estimated is not limited by the number of observations, as it happens with several frequentist approaches. However, it is important to point out that the Bayesian analysis requires informative priors in order for the parameters to be identifiable. The method can be easily extended for the analysis of other medical data sets.63346Begg, C.B., Greenes, R.A., Assessment of diagnostic tests when disease verification is subject to selection bias (1983) Biometrics, 39, pp. 207-215Zhou, X., Maximum likelihood estimators of sensitivity and specificity corrected for verification bias (1983) Commun Statis Theory Meth, 22, pp. 3177-3198Hui, S.L., Walter, S.D., Estimating the error rates of diagnostic tests (1980) Biometrics, 36, pp. 167-171Joseph, L., Gyorkos, T.W., Coupal, L., Bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard (1985) Am J Epidemiol, 141, pp. 263-272Hitt, E., Cancer in the Americas (2003) Lancet Oncol, 4, p. 9Brasil. Ministério da Saúde. Secretaria Nacional de Assistência à Saúde. Instituto Nacional do Câncer. Estimativas da incidência e mortalidade por câncer no Brasil. Rio de Janeiro: INCA2002. Available on website: 〈http://www.inca.org.br/cancer/ epide miologia/estimativa2002/estimativas.html〉Mitchell, M.F., Schottenfeld, D., Tortolero-Luna, G., Cantor, S.B., Richards-Kortum R. Colposcopy for the diagnosis of squamous intraepithelial lesions: A meta-analysis (1998) Obstet Gynecol, 91, pp. 626-631Hopman, E.H., Kenemans, P., Helmerhorst, T.J., Positive predictive rate of colposcopic examination of the cervix uteri: An overview of literature (1998) Obstet Gynecol Surv, 53, pp. 97-106Begg, C.B., Biases in the assessment of diagnostic tests (1987) Stat Med, 6, pp. 411-423Hui, S.L., Zhou, X.H., Evaluation of diagnostic tests without gold standards (1998) Stat Methods Med Res, 7, pp. 354-370Zhou, X.H., Correcting for verification bias in studies of a diagnostic test's accuracy (1998) Stat Methods Med Res, 7, pp. 337-353McCrory, D.C., Matchar, D.B., Bastian, L. et al. 1999. Evaluation of cervical cytology. Evidence report/technology assessment n.5. (Prepared by Duke University under Contract n. 290-97-0014). AHCPR publication n. 99-E010. Rockville: Agency for Health Care Policy and ResearchDendukuri, N., Joseph, L., Bayesian approaches to modelling the conditional dependence between multiple diagnostic tests (2001) Biometrics, 57, pp. 208-217Faraone, S.V., Tsuang, M.T., Measuring diagnostic accuracy in the absence of a "gold standard (1994) Am J Psychiatry, 151, pp. 650-657Qu, Y., Tan, M., Kutner, M.H., Random effects models in latent class analysis for evaluating accuracy of diagnostic tests (1996) Biometrics, 52, pp. 797-810Hadgu, A., Qu, Y., A biomedical application of latent models with random effects (1998) Appl Statist, 47, pp. 603-616Tanner, M., Wong, W., The calculation of posterior distributions by data augmentation (1987) J Am Statist Ass, 82, pp. 528-550Gelfand, A.E., Smith, A.F.M., Sampling Based Approaches to Calculating Marginal Densities (1990) J Am Stat Assoc, 85, pp. 398-409Gelfand, A.E., Gibbs sampling (2000) J Am Stat Assoc, 95, pp. 1300-1304Syrjanen, K., Naud, P., Derchain, S., Comparing PAP smear cytology, aided visual inspection, screening colposcopy, cervicography and HPV testing as optional screening tools in Latin America. Study design and baseline data of the LAMS study (2005) Anticancer Res, 25, pp. 3469-3480Sarian, L.O., Derchain, S.F., Naud, P., Evaluation of visual inspection with acetic acid (VIA), Lugol's iodine (VILI), cervical cytology and HPV testing as cervical screening tools in Latin America. This report refers to partial results from the LAMS (Latin AMerican Screening) study (2005) J Med Screen, 12, pp. 142-149Solomon, D., Davey, D., Kurman, R., The 2001 Bethesda System: Terminology for reporting results of cervical cytology (2002) JAMA, 287, pp. 2114-2119Blumenthal, P., Sanghvi, H., (1997) Atlas for unaided visual inspection of the cervix, , Baltimore and Harare: JHPIEGO Corporation and University of Zimbabwe Medical SchoolNanda, K., McCrory, D.C., Myers, E.R., Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: A systematic review (2000) Ann Intern Med, 132, pp. 810-819Belinson, J.L., Pretorius, R.G., Zhang, W.H., Wu, L.Y., Qiao, Y.L., Elson, P., Cervical cancer screening by simple visual inspection after acetic acid (2001) Obstet Gynecol, 98, pp. 441-444Visual inspection with acetic acid for cervical-cancer screening: Test qualities in a primary care setting (1999) Lancet, 353, pp. 869-873. , University of Zimbabwe/JHPIEGO Cervical Cancer ProjectSchiffman, M., Herrero, R., Hildensheim, A., HPV DNA testing in cervical cancer screening: Results from women in a high-risk province of Costa Rica (2000) JAMA, 283, pp. 87-93Wright Jr, T.C., Lynette, D., Kuhn, L., Pollack, A., Lorincz, A., HPV DNA testing of self-collected vaginal samples compared with cytologic screening to detect cervical cancer (2000) JAMA, 283, pp. 81-86Box, G.E.P., Tiao, G.C., (1992) Bayesian Inference in Statistical Analysis, , Reprint edition. New York: Wiley-InterscienceAltman, D.G., Bland, J.M., Diagnostic tests 2: Predictive values (1994) BMJ, 309, p. 102Franco, E.L., Primary screening of cervical cancer with human papillomavirus tests (2003) J Natl Cancer Inst Monogr, 31, pp. 89-96Geweke J. 1992. Evaluating the accuracy of sampling-based approaches to calculating posterior moments. Bayesian Statistics 4Bernardo, J.M.Berger, J.O.Dawid, A.P and.Smith, A.F.M., Eds.Clarendom Press: Oxford, 169-94Carlin, B.P., Louis, T.A., (2000) Bayes and empirical Bayes methods for data analysis, , 2nd ed. London: Chapman and Hall/CRCKoss, L.G., Human papillomavirus testing as a screening tool for cervical cancer (2000) JAMA, 283, p. 2525Shlay, J.C., Dunn, T., Byers, T., Barón, A.E., Douglas, J.M., Prediction of cervical intraepithelial neoplasia grade 2-3 using risk assessment and human papillomavirus testing in women with atypia on Papanicolaou Smears (2000) Obstet Gynecol, 96, pp. 410-416Spiegelhalter, D.J., Best, N.G., Carlin, B.P., van der Linde A Bayesian measures of model complexity and fit (with discussion) (2002) J Roy Statist Soc B, 64, pp. 583-640Franco, E.L., Ferenczy, A., Assessing gains in diagnostic utility when human papillomavirus testing is used as an adjunct to papanicolaou smear in the triage of women with cervical cytologic abnormalities (1999) Am J Obstet Gynecol, 181, pp. 382-386Macaskill, P., Walter, S.D., Irwig, L., Franco, E.L., Assessing the gain in diagnostic performance when combining two diagnostic tests (2002) Statis Med, 21, pp. 2527-2546Brenner, H., How independent are multiple "independent" diagnostic classifications? Stat MedEspeland, M.A., Handelman, S.L., Using latent class models to characterize and assess relative error in discrete measurements (1989) Biometrics, 45, pp. 587-599Yang, I., Becker, M.P., Latent variable modeling of diagnostic accuracy (1997) Biometrics, 53, pp. 948-958Ratman, S., Franco, E.L., Ferenczy, A., Human papillomavirus testing for primary screening of cervical cancer precursors (2000) Cancer Epidemiol Biomarkers Prev, 9, pp. 945-951Coste, J., Cochand-Priollet, B., de Cremoux, P., Cross sectional study of conventional cervical smear, monolayer cytology and human papillomavirus DNA testing for cervical cancer screening (2003) BMJ, 326, p. 733Schiffman, M., Hildesheim, A., Herrero, R., Bratti, M., In reply: Human papillomavirus testing as a screening tool for cervical cancer (2000) JAMA, 283, pp. 2525-2526Londhe, M., George, S.S., Seshadri, L., Detection of CIN by naked eye visualization after application of acetic acid (1997) Indian J Cancer, 34, pp. 88-91Sankaranarayanan, R., Wesley, R., Somanathan, T., Visual inspection of the uterine cervix after the application of acetic acid in the detection of cervical carcinoma and its precursors (1998) Cancer, 83, pp. 2150-2156Sankaranarayanan, R., Shyamalayumary, B., Wesley, R., Sreedevi Amma, N., Parkin, D.M., Nair, M.K., Visual inspection with acetic acid in the early detection of cervical cancer and precursors (1999) Int J Cancer, 80, pp. 161-163Denny, L., Kuhn, L., Pollack, A., Wainwright, H., Wright Jr., T.C., Evaluation of alternative methods of cervical cancer screening in resource-poor settings (2000) Cancer, 89, pp. 826-833Denny, L., Kuhn, L., Pollack, A., Wright Jr., T.C., Direct visual inspection for cervical cancer screening: An analysis of factors influencing test performance (2002) Cancer, 94, pp. 1699-1707Parmigiani, G., Measuring uncertainty in complex decision analysis models (2002) Stat Methods Med Res, 11, pp. 513-537Koss, L.G., Human papillomavirus testing as a screening tool for cervical cancer (2000) JAMA, 283, pp. 2525-2526Smith, A.F.M., Roberts, G.O., Bayesian Computation via the Gibbs Sampler and Related MCMC Methods (1993) J R Stat SocB, 55, pp. 3-2

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

A computational model of liver iron metabolism

Iron is essential for all known life due to its redox properties; however, these same properties can also lead to its toxicity in overload through the production of reactive oxygen species. Robust systemic and cellular control are required to maintain safe levels of iron, and the liver seems to be where this regulation is mainly located. Iron misregulation is implicated in many diseases, and as our understanding of iron metabolism improves, the list of iron-related disorders grows. Recent developments have resulted in greater knowledge of the fate of iron in the body and have led to a detailed map of its metabolism; however, a quantitative understanding at the systems level of how its components interact to produce tight regulation remains elusive. A mechanistic computational model of human liver iron metabolism, which includes the core regulatory components, is presented here. It was constructed based on known mechanisms of regulation and on their kinetic properties, obtained from several publications. The model was then quantitatively validated by comparing its results with previously published physiological data, and it is able to reproduce multiple experimental findings. A time course simulation following an oral dose of iron was compared to a clinical time course study and the simulation was found to recreate the dynamics and time scale of the systems response to iron challenge. A disease state simulation of haemochromatosis was created by altering a single reaction parameter that mimics a human haemochromatosis gene (HFE) mutation. The simulation provides a quantitative understanding of the liver iron overload that arises in this disease. This model supports and supplements understanding of the role of the liver as an iron sensor and provides a framework for further modelling, including simulations to identify valuable drug targets and design of experiments to improve further our knowledge of this system

Editors’ Introduction: An Overview of the Educational Administration and Leadership Curriculum: Traditions of Islamic Educational Administration and Leadership in Higher Education

This chapter provides an overview of several topics relevant to constructing an approach to teaching educational administration and leadership in Muslim countries. First, it places the topic in the context of the changing nature and critiques of the field that argue for a greater internationalisation to both resist some of the negative aspects of globalisation and to represent countries’ traditions in the professional curriculum. Then, it identifies literature that presents the underlying principles and values of Islamic education that guide curriculum and pedagogy and shape its administration and leadership including the Qur’an and Sunnah and the classical educational literature which focuses on aims, values and goals of education as well as character development upon which a ‘good’ society is built. This is followed by a section on the Islamic administration and leadership traditions that are relevant to education, including the values of educational organisations and how they should be administered, identifying literature on the distinctive Islamic traditions of leadership and administrator education and training as it applies to education from the establishment of Islam and early classical scholars and senior administrators in the medieval period who laid a strong foundation for a highly sophisticated preparation and practice of administration in philosophical writings and the Mirrors of Princes writings, and subsequent authors who have built upon it up to the contemporary period. The final section provides an overview of the chapters in this collection