Parameterization Models for Traceable Characterization of Planar CPW SOL Calibration Standards

International audienceMethods for traceable characterization and uncertainty evaluation of planar 1-port CPW short-open-load (SOL) devices are developed. The agreement of modelling and verification measurement results greatly supports the application of the proposed parameterization models and used FEM-based EM modelling of CPW structures for traceable characterization of planar CPW-based SOL devices in the frequency range between a few kHz up to 50 GHz

Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv

Aims: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. Methods and Results: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. Conclusions: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. Significance and Impact of the Study: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial ‘lipidome’

Test Characteristics of Urinary Lipoarabinomannan and Predictors of Mortality among Hospitalized HIV-Infected Tuberculosis Suspects in Tanzania.

Tuberculosis is the most common cause of death among patients with HIV infection living in tuberculosis endemic countries, but many cases are not diagnosed pre-mortem. We assessed the test characteristics of urinary lipoarabinomannan (LAM) and predictors of mortality among HIV-associated tuberculosis suspects in Tanzania. We prospectively enrolled hospitalized HIV-infected patients in Dar es Salaam, with ≥2 weeks of cough or fever, or weight loss. Subjects gave 2 mLs of urine to test for LAM using a commercially available ELISA, ≥2 sputum specimens for concentrated AFB smear and solid media culture, and 40 mLs of blood for culture. Among 212 evaluable subjects, 143 (68%) were female; mean age was 36 years; and the median CD4 count 86 cells/mm(3). 69 subjects (33%) had culture confirmation of tuberculosis and 65 (31%) were LAM positive. For 69 cases of sputum or blood culture-confirmed tuberculosis, LAM sensitivity was 65% and specificity 86% compared to 36% and 98% for sputum smear. LAM test characteristics were not different in patients with bacteremia but showed higher sensitivity and lower specificity with decreasing CD4 cell count. Two month mortality was 64 (53%) of 121 with outcomes available. In multivariate analysis there was significant association of mortality with absence of anti-retroviral therapy (p = 0.004) and a trend toward association with a positive urine LAM (p = 0.16). Among culture-negative patients mortality was 9 (75%) of 12 in LAM positive patients and 27 (38%) of 71 in LAM negative patients (p = 0.02). Urine LAM is more sensitive than sputum smear and has utility for the rapid diagnosis of culture-confirmed tuberculosis in this high-risk population. Mortality data raise the possibility that urine LAM may also be a marker for culture-negative tuberculosis

Five new species of Terebellides (Annelida, Polychaeta, Trichobranchidae) from Papua New Guinea (Bismarck and Solomon seas)

Five new species of Terebellides are described from coastal and deep waters of Papua New Guinea, using both morphology and molecular tools (for four species). Terebellides elenae n. sp. is characterized by the absence of both a glandular lateral region on TC3 and papillae on margins of the branchial lamellae and by the presence of partially fused branchial lobes with conspicuous fifth lobe and dorsal rounded projections until TC6. Terebellides fauchaldi n. sp. has a very large glandular lateral region on the third thoracic chaetiger (TC3), a fifth branchial lobe and partially fused branchial lobes, and conspicuous dorsal rounded projections on TC2–6. Terebellides madeep n. sp. is characterized by a thin glandular region on TC3 and by four free branchial lobes. Terebellides oculata n. sp. is one of the only two species in the world to have eyespots. Finally, T. papillosa n. sp. has geniculate chaetae on TC6 and TC7 and bears a large number of papillae. A majority-rule consensus tree using the 16S gene and an identification key for all Terebellides species described from the Central Indo-Pacific region are provided

Confirmation of the exotic status of Marphysa victori Lavesque, Daffe, Bonifácio & Hutchings, 2017 (Annelida) in French waters and synonymy of Marphysa bulla Liu, Hutchings & Kupriyanova, 2018

The bait worm Marphysa victori Lavesque, Daffe, Bonifácio & Hutchings, 2017 was originally described from Arcachon Bay, France. In the original description, the authors suggested that it may have been introduced, although definitive evidence was lacking at the time. In this paper, we confirm that M. victori is an exotic species originating from East Asia, probably a native of China or Japan. This species was most likely introduced into Arcachon Bay in the 1970s with non-native oysters Crassostrea gigas Thunberg, 1793. The Asiatic origin of this species is confirmed based on both morphological and molecular evidence. Comparison of the type specimens of M. victori with those of Marphysa bulla Liu, Hutchings & Kupriyanova, 2018 described from the Yellow Sea, China, showed no morphological or molecular differences and therefore the more recently named M. bulla is relegated to a junior synonym. This is the first example of a large-sized errant invertebrate species being introduced with oysters into Arcachon Bay.Initiative d'excellence de l'Université de Bordeau

Synthesis of some nucleosides derivatives from L- rhamnose with expected biological activity

Practical procedures for production of variously blocked compounds from L-rhamnose have been developed. These compounds are highly useful as indirect β-L-rhamnosyl donors. This approach represents a new method for the synthesis of aromatic nucleoside analogues and the synthesis of (3S, 4S, 5S, 6R) 3, 4, 5-triacetoxy-2-methyl-7,9-diaza-1-oxa-spiro [4,5]decane-10-one-8-thione (7)

Identification of an α(1→6) mannopyranosyltransferase (MptA), involved in Corynebacterium glutamicum lipomanann biosynthesis, and identification of its orthologue in Mycobacterium tuberculosis

Corynebacterium glutamicum and Mycobacterium tuberculosis share a similar cell wall architecture, and the availability of their genome sequences has enabled the utilization of C. glutamicum as a model for the identification and study of, otherwise essential, mycobacterial genes involved in lipomannan (LM) and lipoarabinomannan (LAM) biosynthesis. We selected the putative glycosyltransferase-Rv2174 from M. tuberculosis and deleted its orthologue NCgl2093 from C. glutamicum. This resulted in the formation of a novel truncated lipomannan (Cg-t-LM) and a complete ablation of LM/LAM biosynthesis. Purification and characterization of Cg-t-LM revealed an overall decrease in molecular mass, a reduction of α(1→6) and α(1→2) glycosidic linkages illustrating a reduced degree of branching compared with wild-type LM. The deletion mutant's biochemical phenotype was fully complemented by either NCgl2093 or Rv2174. Furthermore, the use of a synthetic neoglycolipid acceptor in an in vitro cell-free assay utilizing the sugar donor β-d-mannopyranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor α-d-Manp-(1→6)-α-d-Manp-O-C8 as a substrate, confirmed NCgl2093 and Rv2174 as an α(1→6) mannopyranosyltransferase (MptA), involved in the latter stages of the biosynthesis of the α(1→6) mannan core of LM. Altogether, these studies have identified a new mannosyltransferase, MptA, and they shed further light on the biosynthesis of LM/LAM in Corynebacterianeae

The Mycobacterium Tuberculosis FAS-II Dehydratases and Methyltransferases Define the Specificity of the Mycolic Acid Elongation Complexes

BACKGROUND: The human pathogen Mycobacterium tuberculosis (Mtb) has the originality of possessing a multifunctional mega-enzyme FAS-I (Fatty Acid Synthase-I), together with a multi-protein FAS-II system, to carry out the biosynthesis of common and of specific long chain fatty acids: the mycolic acids (MA). MA are the main constituents of the external mycomembrane that represents a tight permeability barrier involved in the pathogenicity of Mtb. The MA biosynthesis pathway is essential and contains targets for efficient antibiotics. We have demonstrated previously that proteins of FAS-II interact specifically to form specialized and interconnected complexes. This finding suggested that the organization of FAS-II resemble to the architecture of multifunctional mega-enzyme like the mammalian mFAS-I, which is devoted to the fatty acid biosynthesis. PRINCIPAL FINDINGS: Based on conventional and reliable studies using yeast-two hybrid, yeast-three-hybrid and in vitro Co-immunoprecipitation, we completed here the analysis of the composition and architecture of the interactome between the known components of the Mtb FAS-II complexes. We showed that the recently identified dehydratases HadAB and HadBC are part of the FAS-II elongation complexes and may represent a specific link between the core of FAS-II and the condensing enzymes of the system. By testing four additional methyltransferases involved in the biosynthesis of mycolic acids, we demonstrated that they display specific interactions with each type of complexes suggesting their coordinated action during MA elongation. SIGNIFICANCE: These results provide a global update of the architecture and organization of a FAS-II system. The FAS-II system of Mtb is organized in specialized interconnected complexes and the specificity of each elongation complex is given by preferential interactions between condensing enzymes and dehydratase heterodimers. This study will probably allow defining essential and specific interactions that correspond to promising targets for Mtb FAS-II inhibitors

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment