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Campbell K.,University of Cambridge | Vowinckel J.,University of Cambridge | Keller M.A.,University of Cambridge | Ralser M.,University of Cambridge | Ralser M.,Francis Crick Institute Mill Hill Laboratory
Antioxidants and Redox Signaling

Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway. Antioxid. Redox Signal. 24, 543-547. © Kate Campbell et al., 2016. Source

Thavayogarajah T.,University of Marburg | Gangopadhyay P.,University of Marburg | Rahlfs S.,Justus Liebig University | Becker K.,Justus Liebig University | And 3 more authors.

Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. © 2015 Thavayogarajah et al. Source

Diedrich C.R.,University of Cape Town | O'Hern J.,University of Cape Town | Wilkinson R.J.,University of Cape Town | Wilkinson R.J.,Francis Crick Institute Mill Hill Laboratory | Wilkinson R.J.,Imperial College London

Summary Infection with HIV-1 greatly increases the risk of active tuberculosis (TB). Although hypotheses suggest HIV-1 disrupts Mycobacterium tuberculosis (Mtb) granuloma function, few studies have examined this directly. The objective of this study was to determine what evidence exists about the effect HIV-1 co-infection has upon Mtb granulomas. A systematic search of PubMed, Web of Science, and Medline up to 20 March 2015 was conducted, to identify studies comparing Mtb-infected tissue from HIV-1 infected and uninfected persons, or HIV-1 infected persons with stratified peripheral CD4 T cell (pCD4) counts. We summarized findings that focused on how HIV-1 changes granuloma formation, bacterial presence, cellular composition, and cytokine production. Nineteen studies with a combined sample size of 899 persons were included. Although studies frequently were limited by variable or inadequately described definitions of outcomes and analytical methods, HIV-1 was found to be associated with increased bacillary load within Mtb-infected tissue. Reductions in pCD4 counts within co-infected persons associated with both poorer granuloma formation and higher bacterial load. The high degree of heterogeneity among studies combined with experimental limitations made it difficult to conclusively support previously published and prevalent hypotheses about HIV-1/Mtb co-infection granulomas. To elucidate the validity of these hypotheses we have described areas that can be improved in future studies in order to clarify the influence HIV-1 co-infection has upon the Mtb granuloma. © 2016 Elsevier Ltd. Source

Sumner T.,London School of Hygiene and Tropical Medicine | Houben R.M.G.J.,London School of Hygiene and Tropical Medicine | Rangaka M.X.,University College London | Rangaka M.X.,University of Cape Town | And 7 more authors.

Background: In HIV-uninfected individuals, isoniazid preventive therapy (IPT) has been associated with long-term protection against tuberculosis (TB). For HIV-infected/antiretroviral therapy (ART)-naive individuals, high TB rates have been observed following completion of IPT, consistent with a lack of 'cure' of infection. Recent trial data of IPT among HIV-infected individuals on ART in Khayelitsha, South Africa, have suggested that the effect of IPT persisted following completion of IPT. Methods: Using mathematical modelling, we explored if this increased duration of protection may be due to an increased curative ability of IPT when given in combination with ART. The model was used to estimate the annual risk of infection and proportion of individuals whose latent infection was 'cured' by IPT, defined such that they must be reinfected to be at risk of disease. Results: The estimated annual risk of infection was 4.0% (2.6-5.8) and the estimated proportion of individuals whose latent Mycobacterium tuberculosis infection was cured following IPT was 35.4% (2.4-76.4), higher than that previously estimated for HIV-infected/ART-naive individuals. Our results suggest that IPT can cure latent M. tuberculosis infection in approximately one-third of HIV-infected individuals on ART and therefore provide protection beyond the period of treatment. Conclusion: Among HIV-infected individuals on ART in low incidence settings, 12 months of IPT may provide additional long-term benefit. Among HIV-infected individuals on ART in high incidence settings, the durability of this protection will be limited because of continued risk of reinfection, and continuous preventive therapy together with improved infection control efforts will be required to provide long-term protection against TB. © 2016 Wolters Kluwer Health, Inc. All rights reserved. Source

Bahr N.C.,University of Minnesota | Marais S.,University of Cape Town | Van Crevel R.,Radboud University Nijmegen | Wilkinson R.J.,University of Cape Town | And 5 more authors.
Clinical Infectious Diseases

Tuberculous meningitis (TBM) is the most severe form of tuberculous with substantial mortality. In May 2015, 54 researchers from 10 countries met in Da Lat, Vietnam, to discuss advances in TBM. Among the attendees were researchers involved in pivotal studies on the use of Xpert MTB/Rif for TBM diagnosis. Attendees discussed the 2014World Health Organization strong recommendation favoring the use of Xpert "in preference to conventional microscopy and culture as the initial diagnostic test for cerebrospinal fluid (CSF) if the sample volume is low or if additional specimens cannot be obtained to make a quick diagnosis." Attendees were concerned that the limitations of Xpert testing for TBM are not emphasized. Clear guidance is needed for the investigational pathway for TBM, including recommendations on the diagnostic package of investigations, which does not stop with Xpert testing. Second, emphasis on the large CSF volumes (ideally 8-10 mL) needed for Xpert testing is required. Guidelines should also emphasize that TBM is a medical emergency and early treatment reduces mortality. © 2016 The Author. Source

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