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  • 99
    ATCC mycobacterium bovis bcg
    Di- O -acyl trehalose interferes with the maturation of DCs activated with <t>BCG</t> antigens. DC bone marrow precursors cultured with GM-CSF for six days were incubated with 20 μg M . <t>bovis</t> /BCG cell wall antigens for 24 h; the maturation markers were analyzed by FACS in CD11c + cells (A). Increased expression of MHC-I, MHC-II and costimulatory molecules CD40, CD80 and CD86 are observed (p
    Mycobacterium Bovis Bcg, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bcg  (ATCC)
    99
    ATCC bcg
    Effect of lipid antigens of Mycobacterium <t>bovis</t> <t>BCG</t> vaccine and TLR-activation on the ability of mouse macrophages to process and present a peptide antigen 85B derived epitope from BCG to T cells
    Bcg, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bcg tice
    <t>Bioxy</t> vs. Mycobacterium <t>bovis</t>
    Bcg Tice, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bcg montreal
    <t>DosR</t> Asp-54 is required for gene induction but not for binding. EMSA of mutant DosR (Asp-54Glu) with labelled acr promoter DNA. Lanes: 1, no extract; 2, induced extract of DosR (Asp-54Glu); 3, induced extract of DosR (WT). Hypoxic induction of acr by mutant DosR (Asp-54Glu). Acr expression measured by luciferase reporter gene in <t>BCG</t> and in BCG with DosR (Asp-54Glu) replacing DosR (WT). Shown are representative data from one of two experiments, each of which was performed in duplicate.
    Bcg Montreal, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC mycobacterium bovis calmette guérin bcg
    Linear regression curves, R 2 values, and confidence and prediction intervals (95%) corresponding to devR , IS 1311 , and ITSdenak probe C T values in spiked samples with different concentrations of M. <t>bovis</t> <t>BCG</t> (A), M. avium subsp. hominissuis (B), or M.
    Mycobacterium Bovis Calmette Guérin Bcg, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATCC bcg phipps
    Linear regression curves, R 2 values, and confidence and prediction intervals (95%) corresponding to devR , IS 1311 , and ITSdenak probe C T values in spiked samples with different concentrations of M. <t>bovis</t> <t>BCG</t> (A), M. avium subsp. hominissuis (B), or M.
    Bcg Phipps, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATCC bcg birkhaug
    Linear regression curves, R 2 values, and confidence and prediction intervals (95%) corresponding to devR , IS 1311 , and ITSdenak probe C T values in spiked samples with different concentrations of M. <t>bovis</t> <t>BCG</t> (A), M. avium subsp. hominissuis (B), or M.
    Bcg Birkhaug, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bcg moreau
    <t>BCG</t> and M. <t>brumae</t> survival inside T24, MB49 and 5637 BC cells. Colony-forming units (CFUs) from cell lysates at different time-points after infection are represented (h, hours). Values represent the means ± SEMs of three serial dilutions of triplicate culture wells of M. brumae infected T24 cells ( a ), MB49 cells ( b ) and 5637 cells ( c ), and BCG-infected T24 cells ( a ). Data are representative of one out of three (for T24) and two (for MB49 and 5637) independent experiments. BCG counts were estimated from the results obtained in the 96-well plate 7H10-based spot assay from dilutions of infected MB49 and 5637 BC cells.
    Bcg Moreau, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC mycobacterium bcg
    TLR2-mediated CCL5 production by epithelial cells involves calcineurin. HEK and TLR2 cells were pretreated with FK-506 (100 ng/ml) for 1 h. After incubation, cells were either left uninfected or stimulated with M. <t>bovis</t> <t>BCG</t> for a further 24 h. Culture
    Mycobacterium Bcg, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bcg russia
    Bacterial growth in lungs (A and B) and spleens (C and D) after low-dose aerosol infection of 6–8-week-old C57BL/6 mice with wild-type <t>H37Rv</t> ([unk]), H37Rv:ΔRD1 (■), or <t>BCG-Russia</t> (▲). A and C, Early-phase infection and dissemination. B and D, Progression of infection through 21 weeks. Data at each time point are the mean and SD of 15 mice per strain from 3 separate infections.
    Bcg Russia, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bcg strain
    Metabolic network of the central metabolism of Mycobacterium <t>bovis</t> <t>BCG.</t> Glycolysis/gluconeogenesis (green), oxidative pentose phosphate pathway (orange), tricarboxylic acid cycle (TCA, pink), anaplerotic reactions (blue), and biosynthesis (grey). Standard abbreviations are used for the amino acids. Metabolite abbreviations: ACCOA, acetyl-CoA; ACE, acetate; CHO, chorismate; E4P, d -erythrose 4-phosphate; F6P, d -fructose 6-phosphate; FBP, d -fructose 1,6-bisphosphate; FUM, fumarate; G6P, d -glucose 6-phosphate; GA3P, glyceraldehyde 3-phosphate; GLX, glyoxylate; GLYC, glycerol; ICIT, isocitrate/citrate; KIV, 2-oxoisovalerate; MALOAA, l -malate-oxaloacetate; OXG, 2-oxoglutarate; R5P,α- d -ribose 5-phosphate/ l -ribulose 5-phosphate/ l -xylulose 5-phosphate; PEP, phosphoenolpyruvate; PGA, 2-phospho- d -glycerate/3-phospho- d -glycerate; PYR, pyruvate; S7P, sedoheptulose 7-phosphate; SUC, succinate; SUCCOA, succinyl-CoA. Enzyme abbreviations: CS: citrate synthase; ENO: enolase; FBA: fructose-bisphosphate adolase; FBP: fructose-bisphosphatase; FUM:fumurase; GAPA: glyceraldehyde 3-phosphate dehydrogenase; GND: 6-phosphogluconate dehydrogenase; ICL: isocitrate lyase; ICDH: isocitrate dehydrogenase NADP-dependent; KOR/KGD: α-ketoglutarate ferredoxin oxidoreductase/α-ketoglutarate decarboxylase;MEZ/PCA: malic enzyme/pyruvate carboxylase: MDH: malate dehydrogenase; MS: malate synthase; PCK: phosphoenolpyruvate carboxykinase; PDH: pyruvate dehydrogenase; PGI: glucose phosphate isomerase; PYK: pyruvate kinase;SDH: succinate dehydrogenase; SCS: Succinyl CoA synthetase; TAL: transaldolase; TKT1/2: transketolase. Explicit names are also given in Table S6 . The picture was generated with Omix, an editor for biochemical networks and visualization tool [ http://www.13cflux.net/omix ].
    Bcg Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC bcg tokyo 172 growth mycobacterium bovis bcg tokyo 172
    Functional characterization of significantly altered proteins in THP-1 cells infected with MTBC strain <t>H37Rv</t> , M. bovis , or <t>BCG.</t> (A) Top canonical pathways, (B) diseases and disorders, (C) molecular and cellular functions, (D) physiological system development and functions, and (E) toxicity functions.
    Bcg Tokyo 172 Growth Mycobacterium Bovis Bcg Tokyo 172, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATCC bcg atcc 35736
    Functional characterization of significantly altered proteins in THP-1 cells infected with MTBC strain <t>H37Rv</t> , M. bovis , or <t>BCG.</t> (A) Top canonical pathways, (B) diseases and disorders, (C) molecular and cellular functions, (D) physiological system development and functions, and (E) toxicity functions.
    Bcg Atcc 35736, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC avirulent strain mycobacterium bovis bcg pasteur
    Survival curves of mice infected with M. tuberculosis or M. <t>bovis</t> <t>BCG</t> Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.
    Avirulent Strain Mycobacterium Bovis Bcg Pasteur, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATCC m bovis bcg atcc 35747
    Survival curves of mice infected with M. tuberculosis or M. <t>bovis</t> <t>BCG</t> Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.
    M Bovis Bcg Atcc 35747, supplied by ATCC, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Connaught Labs infectious dose mycobacterium bovis bcg
    Survival curves of mice infected with M. tuberculosis or M. <t>bovis</t> <t>BCG</t> Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.
    Infectious Dose Mycobacterium Bovis Bcg, supplied by Connaught Labs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Connaught Labs mycobacterial preparation mycobacterium bovis bcg
    Survival curves of mice infected with M. tuberculosis or M. <t>bovis</t> <t>BCG</t> Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.
    Mycobacterial Preparation Mycobacterium Bovis Bcg, supplied by Connaught Labs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC mycobacterium bovis type strain
    Survival curves of mice infected with M. tuberculosis or M. <t>bovis</t> <t>BCG</t> Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.
    Mycobacterium Bovis Type Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Prestwick Chemical mycobacterium bovis bcg
    Survival curves of mice infected with M. tuberculosis or M. <t>bovis</t> <t>BCG</t> Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.
    Mycobacterium Bovis Bcg, supplied by Prestwick Chemical, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore bcg
    Combined therapy of <t>BCG</t> and L-NAME exerts an antitumoral effect through modulation of MACs and fibroblasts in the tumor microenvironment. BCG directly induces fibroblast proliferation via MAPK and <t>PI3K</t> signaling pathways as well as alpha-SMA and collagen expression. BCG induces in MACs the production of NO and soluble factors including FGF-2 which induce fibroblast proliferation. BCG therapy increases collagen deposition and expression of alpha-SMA and FGF-2 in bladder tumors The treatment with L-NAME, improves the stimulation of fibroblasts by BCG.
    Bcg, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Pasteur Institute bcg
    ). The heavy arrows indicate methyltransferase open reading frames. Small arrows indicate primers used to amplify the 5′ and 3′ regions of mma3 . Thin solid lines represent the PCR products. Open squares, Sal I sites. Shown below the genomic regions are the amplicons from wild-type and mutant mma3 5′ regions, with Sal I sites indicated (note the additional site in the mutant mma3 ). Scale marker, 1,000 bp. (B) Purified 562-bp PCR products containing the 5′ end of the mma3 gene from the 13 <t>BCG</t> strains were digested with Sal I. Without digestion, a 562-bp product is seen. After incubation, the wild-type strains have three fragments of 303, 144, and 115 bp, while the mutant strains have four fragments of 246, 144, 115, and 57 bp. (C) TLC analysis of purified mycolic acids from various BCG substrains. The same BCG strains shown in the PCR-RFLP gel were analyzed by TLC for production of mycolic acids as described in Materials and Methods. The three bands obtained represent (from bottom to top) ketomycolates, methoxymycolates, and α-mycolates. It is seen in this figure that eight BCG strains lack methoxymycolates. These are the same strains that gave the extra Sal I band; they represent strains of BCG obtained from the Pasteur Institute in <t>1931</t> or later.
    Bcg, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 93/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Di- O -acyl trehalose interferes with the maturation of DCs activated with BCG antigens. DC bone marrow precursors cultured with GM-CSF for six days were incubated with 20 μg M . bovis /BCG cell wall antigens for 24 h; the maturation markers were analyzed by FACS in CD11c + cells (A). Increased expression of MHC-I, MHC-II and costimulatory molecules CD40, CD80 and CD86 are observed (p

    Journal: PLoS ONE

    Article Title: Mycobacterial glycolipid Di-O-acyl trehalose promotes a tolerogenic profile in dendritic cells

    doi: 10.1371/journal.pone.0207202

    Figure Lengend Snippet: Di- O -acyl trehalose interferes with the maturation of DCs activated with BCG antigens. DC bone marrow precursors cultured with GM-CSF for six days were incubated with 20 μg M . bovis /BCG cell wall antigens for 24 h; the maturation markers were analyzed by FACS in CD11c + cells (A). Increased expression of MHC-I, MHC-II and costimulatory molecules CD40, CD80 and CD86 are observed (p

    Article Snippet: Culture of Mycobacterium bovis /BCG and isolation of cell walls Mycobacteria were obtained from the American Type Culture Collection (ATCC 35733) and growth for 14 days in Sauton medium.

    Techniques: Cell Culture, Incubation, FACS, Expressing

    Di- O -acyl trehalose promotes the expansion of regulatory T lymphocytes. We studied the capacity of DCs activated with M . bovis /BCG antigens and TLR agonists with or without preincubation with DAT. DCs were cocultured with lymphocytes CD4 + CD25 - FoxP3 - and CD4 + CD25 + FoxP3 + lymphocytes obtained by cell sorting from Foxp3GFP + transgenic mice. The proliferation was measured by a dilution method with eDye Fluor 670 (A). Proliferation of FoxP3 - lymphocytes was observed in cocultures with DCs activated with DAT, BCG antigens, and LPS (p

    Journal: PLoS ONE

    Article Title: Mycobacterial glycolipid Di-O-acyl trehalose promotes a tolerogenic profile in dendritic cells

    doi: 10.1371/journal.pone.0207202

    Figure Lengend Snippet: Di- O -acyl trehalose promotes the expansion of regulatory T lymphocytes. We studied the capacity of DCs activated with M . bovis /BCG antigens and TLR agonists with or without preincubation with DAT. DCs were cocultured with lymphocytes CD4 + CD25 - FoxP3 - and CD4 + CD25 + FoxP3 + lymphocytes obtained by cell sorting from Foxp3GFP + transgenic mice. The proliferation was measured by a dilution method with eDye Fluor 670 (A). Proliferation of FoxP3 - lymphocytes was observed in cocultures with DCs activated with DAT, BCG antigens, and LPS (p

    Article Snippet: Culture of Mycobacterium bovis /BCG and isolation of cell walls Mycobacteria were obtained from the American Type Culture Collection (ATCC 35733) and growth for 14 days in Sauton medium.

    Techniques: FACS, Transgenic Assay, Mouse Assay

    Effect of l -methionine- S -sulfoximine (MS) on the growth of various bacteria in broth culture. The bacterial strains indicated were inoculated into an appropriate broth at an initial cell density of 10 5 cells/ml, except M . avium (5 × 10 5 cells/ml) and L. pneumophila and E . coli (10 6 cells/ ml). Mycobacteria were cultured in supplemented 7H9 medium, L. pneumophila in yeast extract medium, and E . coli in Luria-Bertani medium. All bacterial cultures were grown in the presence of various concentrations of l -methionine- S -sulfoximine, as indicated, at 37°C and, except for E . coli , in a 5% CO 2 atmosphere. M . tuberculosis , M . bovis , and M . avium were grown for 4 wk; M . smegmatis and M . phlei for 3 d; L . pneumophila for 24 h; and E . coli for 6 h. For the enumeration of viable bacteria in each culture, aliquots were removed weekly ( M . tuberculosis , M . bovis , and M . avium ), daily ( M . smegmatis and M . phlei ), every 4 h ( L . pneumophila ), or hourly ( E . coli ), plated on solid medium, and incubated at 37°C in a 5% CO 2 atmosphere for 2 wk ( M . tuberculosis , M . bovis , and M . avium ), 3 d ( M . smegmatis and M . phlei ), 24 h ( L . pneumophila ), or 12 h ( E . coli ). All cultures were in duplicate. For each time point, the variation in the colony count averaged ∼5% and never exceeded 10%. (A) M. tuberculosis Erdman; (B) M. tuberculosis H37Rv; (C) M. tuberculosis H37Ra; (D) M. bovis strain 19210; (E) M. bovis strain BCG; (F) M. avium strain 25291; (G) M. smegmatis strain 1-2c; (H) M. phlei strain 11758; (I) L. pneumophila Philadelphia 1; (J) E. coli DH5α.

    Journal: The Journal of Experimental Medicine

    Article Title: An Inhibitor of Exported Mycobacterium tuberculosis Glutamine Synthetase Selectively Blocks the Growth of Pathogenic Mycobacteria in Axenic Culture and in Human Monocytes: Extracellular Proteins as Potential Novel Drug Targets

    doi:

    Figure Lengend Snippet: Effect of l -methionine- S -sulfoximine (MS) on the growth of various bacteria in broth culture. The bacterial strains indicated were inoculated into an appropriate broth at an initial cell density of 10 5 cells/ml, except M . avium (5 × 10 5 cells/ml) and L. pneumophila and E . coli (10 6 cells/ ml). Mycobacteria were cultured in supplemented 7H9 medium, L. pneumophila in yeast extract medium, and E . coli in Luria-Bertani medium. All bacterial cultures were grown in the presence of various concentrations of l -methionine- S -sulfoximine, as indicated, at 37°C and, except for E . coli , in a 5% CO 2 atmosphere. M . tuberculosis , M . bovis , and M . avium were grown for 4 wk; M . smegmatis and M . phlei for 3 d; L . pneumophila for 24 h; and E . coli for 6 h. For the enumeration of viable bacteria in each culture, aliquots were removed weekly ( M . tuberculosis , M . bovis , and M . avium ), daily ( M . smegmatis and M . phlei ), every 4 h ( L . pneumophila ), or hourly ( E . coli ), plated on solid medium, and incubated at 37°C in a 5% CO 2 atmosphere for 2 wk ( M . tuberculosis , M . bovis , and M . avium ), 3 d ( M . smegmatis and M . phlei ), 24 h ( L . pneumophila ), or 12 h ( E . coli ). All cultures were in duplicate. For each time point, the variation in the colony count averaged ∼5% and never exceeded 10%. (A) M. tuberculosis Erdman; (B) M. tuberculosis H37Rv; (C) M. tuberculosis H37Ra; (D) M. bovis strain 19210; (E) M. bovis strain BCG; (F) M. avium strain 25291; (G) M. smegmatis strain 1-2c; (H) M. phlei strain 11758; (I) L. pneumophila Philadelphia 1; (J) E. coli DH5α.

    Article Snippet: E. coli DH5α, L. pneumophila Philadelphia 1, the M. tuberculosis strains Erdman (35801; American Type Culture Collection [ATCC]), H37Rv (25618; ATCC), and H37Ra (25177; ATCC), M. bovis (19210; ATCC), M . bovis BCG (bacille Calmette-Guérin [19274; ATCC]), M. phlei (11758; ATCC), M. smegmatis (14468; ATCC), and Mycobacterium avium (25291; ATCC) were cultured as described ( ).

    Techniques: Mass Spectrometry, Cell Culture, Incubation

    Effect of lipid antigens of Mycobacterium bovis BCG vaccine and TLR-activation on the ability of mouse macrophages to process and present a peptide antigen 85B derived epitope from BCG to T cells

    Journal: Cellular immunology

    Article Title: BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9

    doi: 10.1016/j.cellimm.2013.11.007

    Figure Lengend Snippet: Effect of lipid antigens of Mycobacterium bovis BCG vaccine and TLR-activation on the ability of mouse macrophages to process and present a peptide antigen 85B derived epitope from BCG to T cells

    Article Snippet: They were then infected with Mycobacterium bovis BCG (Pasteur strain, ATCC-35734; MOI=1), prepared as a single cell suspension, for 4 hr on a shaker at 37°C, washed and collected for further analysis.

    Techniques: Activation Assay, Derivative Assay

    CLP DNA vaccination protects mice from TB challenge. Immunization with DNA vaccines expressing the Clp1, 2 and 6 confers partial protection against M. tuberculosis H37Rv infection following aerosol challenge. Parental vector (Cont) and BCG were used as

    Journal:

    Article Title: Immunological diversity within a family of cutinase-like proteins of Mycobacterium tuberculosis

    doi: 10.1016/j.vaccine.2008.05.007

    Figure Lengend Snippet: CLP DNA vaccination protects mice from TB challenge. Immunization with DNA vaccines expressing the Clp1, 2 and 6 confers partial protection against M. tuberculosis H37Rv infection following aerosol challenge. Parental vector (Cont) and BCG were used as

    Article Snippet: With the exception of Clp1, BCG Pasteur (ATCC 35734) encodes all CLP, so their use as homologous boosting antigens may reactivate effective memory immune responses.

    Techniques: Mouse Assay, Expressing, Infection, Plasmid Preparation

    Macrophages infected with pathogenic mycobacteria show decreased MAPK activation and downstream production of inflammatory mediators compared to macrophages infected with nonpathogenic mycobacteria. (A) Western blot analysis of p38 phosphorylation (pp38) following infection with M. avium 724, M. avium 101, M. bovis BCG, and M. phlei . Noninfected macrophages (RC) were treated the same as infected macrophages for all incubations. Blots were stripped and reprobed with a p38 antibody. Densitometry was performed with the 24-h phospho-p38 blot. AU, arbitrary units. (B) NOS2 expression was detected by Western blotting. (C and D) TNF-α and IL-1β present in the culture supernatants at each time point were detected by ELISA. a, P

    Journal: Infection and Immunity

    Article Title: Differential Regulation of the Mitogen-Activated Protein Kinases by Pathogenic and Nonpathogenic Mycobacteria

    doi: 10.1128/IAI.70.6.3040-3052.2002

    Figure Lengend Snippet: Macrophages infected with pathogenic mycobacteria show decreased MAPK activation and downstream production of inflammatory mediators compared to macrophages infected with nonpathogenic mycobacteria. (A) Western blot analysis of p38 phosphorylation (pp38) following infection with M. avium 724, M. avium 101, M. bovis BCG, and M. phlei . Noninfected macrophages (RC) were treated the same as infected macrophages for all incubations. Blots were stripped and reprobed with a p38 antibody. Densitometry was performed with the 24-h phospho-p38 blot. AU, arbitrary units. (B) NOS2 expression was detected by Western blotting. (C and D) TNF-α and IL-1β present in the culture supernatants at each time point were detected by ELISA. a, P

    Article Snippet: Mycobacterium phlei ATCC11758, Mycobacterium smegmatis strain MC2 155 (generously provided by Rich Groger, Washington University, St. Louis, Mo.), and Mycobacterium bovis BCG ATCC 35734 were grown for 4 days ( M. phlei and M. smegmatis ) or 10 days ( M. bovis BCG) as previously described ( ).

    Techniques: Infection, Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    High-throughput screening of the chemical compound library on hypoxic M. bovis BCG MtbNar . (a) Schematic representation of the M. bovis BCG MtbNar hypoxic shift down HTS process. (b) Histogram showing the distribution of compounds versus activity for the HTS. Plate median activity was normalized to an arbitrary value of 1, and the putative hits were identified from those of activity

    Journal: ACS Chemical Biology

    Article Title: A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis

    doi: 10.1021/cb2004884

    Figure Lengend Snippet: High-throughput screening of the chemical compound library on hypoxic M. bovis BCG MtbNar . (a) Schematic representation of the M. bovis BCG MtbNar hypoxic shift down HTS process. (b) Histogram showing the distribution of compounds versus activity for the HTS. Plate median activity was normalized to an arbitrary value of 1, and the putative hits were identified from those of activity

    Article Snippet: Bacterial Strains, Culture Media, and Chemicals M. tuberculosis H37Rv (ATCC 27294), M. bovis BCG Pasteur (ATCC 35734), derived mutant, and complemented strain, were maintained in Dubos broth (Difco) supplemented with 0.05% (v/v) Tween 80 and 10% Dubos Medium Albumin (Difco).

    Techniques: High Throughput Screening Assay, Activity Assay

    Complementation of M. bovis BCG with narGHJI cluster gene from M. tuberculosis enhanced nitrate reductase activity and ATP levels in the hypoxic shift down model. (a) Production of nitrite in culture by M. bovis BCG wild type (WT), M. bovis BCG narGHJI KO strain (BCG nar-KO ), and M. bovis BCG complemented with M. tuberculosis narGHJI (BCG MtbNar ) at days 1 and 5. (b) Intracellular ATP production measured for M. bovis BCG WT and M. bovis BCG MtbNar complemented strains at day 1 and 2 upon methylene blue decolorization in the absence or presence of 20 mM sodium nitrate (NaNO 3 ). Results are expressed as the means ± SD of triplicates.

    Journal: ACS Chemical Biology

    Article Title: A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis

    doi: 10.1021/cb2004884

    Figure Lengend Snippet: Complementation of M. bovis BCG with narGHJI cluster gene from M. tuberculosis enhanced nitrate reductase activity and ATP levels in the hypoxic shift down model. (a) Production of nitrite in culture by M. bovis BCG wild type (WT), M. bovis BCG narGHJI KO strain (BCG nar-KO ), and M. bovis BCG complemented with M. tuberculosis narGHJI (BCG MtbNar ) at days 1 and 5. (b) Intracellular ATP production measured for M. bovis BCG WT and M. bovis BCG MtbNar complemented strains at day 1 and 2 upon methylene blue decolorization in the absence or presence of 20 mM sodium nitrate (NaNO 3 ). Results are expressed as the means ± SD of triplicates.

    Article Snippet: Bacterial Strains, Culture Media, and Chemicals M. tuberculosis H37Rv (ATCC 27294), M. bovis BCG Pasteur (ATCC 35734), derived mutant, and complemented strain, were maintained in Dubos broth (Difco) supplemented with 0.05% (v/v) Tween 80 and 10% Dubos Medium Albumin (Difco).

    Techniques: Activity Assay

    Effect of proton motive force inhibitors, phenothiazines, and standard anti-TB drugs on ATP levels of hypoxic shifted M. bovis BCG MtbNar . Reference compounds at indicated concentrations were tested on hypoxic adapted M. bovis BCG MtbNar complemented strain in 384-well microwell plates (40 μL/well). Hypoxic shift down bacilli were added together with nitrate upon methylene blue decolorization in control plates. After 2 days of drug exposure plates were treated with 40 μL of BTG reagent. ATP levels (RLU) were quantified 10 min after incubation. The experiment was carried out three times in triplicates and results are given as means ± SD. Nig: nigericine; Val: valinomycin; TRZ: thioridazine; MTZ: metronidazole; INH: isoniazid; Rif: rifampicin.

    Journal: ACS Chemical Biology

    Article Title: A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis

    doi: 10.1021/cb2004884

    Figure Lengend Snippet: Effect of proton motive force inhibitors, phenothiazines, and standard anti-TB drugs on ATP levels of hypoxic shifted M. bovis BCG MtbNar . Reference compounds at indicated concentrations were tested on hypoxic adapted M. bovis BCG MtbNar complemented strain in 384-well microwell plates (40 μL/well). Hypoxic shift down bacilli were added together with nitrate upon methylene blue decolorization in control plates. After 2 days of drug exposure plates were treated with 40 μL of BTG reagent. ATP levels (RLU) were quantified 10 min after incubation. The experiment was carried out three times in triplicates and results are given as means ± SD. Nig: nigericine; Val: valinomycin; TRZ: thioridazine; MTZ: metronidazole; INH: isoniazid; Rif: rifampicin.

    Article Snippet: Bacterial Strains, Culture Media, and Chemicals M. tuberculosis H37Rv (ATCC 27294), M. bovis BCG Pasteur (ATCC 35734), derived mutant, and complemented strain, were maintained in Dubos broth (Difco) supplemented with 0.05% (v/v) Tween 80 and 10% Dubos Medium Albumin (Difco).

    Techniques: Incubation

    Viability and intracellular ATP levels of M. bovis BCG during the hypoxic shift down assay. (a) Upper row: A hypoxic M. bovis culture was inoculated at an OD 600 of 0.2 in a 24-well plate and shifted into a hypoxic atmosphere. The presence of oxygen left in the culture was monitored by decolorization of methylene blue. Methylene blue decolorized after 2 days. Lower row: Control wells with methylene blue, but without bacilli, in a hypoxic atmosphere. (b) ATP levels (RLU, relative luminescence units) were monitored daily for a period of 7 days. (c) Survival of M. bovis BCG and M. bovis BCG: ΔdosR in the hypoxic shift down model. Viability (CFU/mL) of M. bovis BCG parental (black bar), M. bovis BCG: ΔdosR (white bar), and complemented mutant (gray bar) was determined at each time point in the hypoxic shift down model. The experiment was carried out three times in triplicate, and results are given as means ± SD.

    Journal: ACS Chemical Biology

    Article Title: A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis

    doi: 10.1021/cb2004884

    Figure Lengend Snippet: Viability and intracellular ATP levels of M. bovis BCG during the hypoxic shift down assay. (a) Upper row: A hypoxic M. bovis culture was inoculated at an OD 600 of 0.2 in a 24-well plate and shifted into a hypoxic atmosphere. The presence of oxygen left in the culture was monitored by decolorization of methylene blue. Methylene blue decolorized after 2 days. Lower row: Control wells with methylene blue, but without bacilli, in a hypoxic atmosphere. (b) ATP levels (RLU, relative luminescence units) were monitored daily for a period of 7 days. (c) Survival of M. bovis BCG and M. bovis BCG: ΔdosR in the hypoxic shift down model. Viability (CFU/mL) of M. bovis BCG parental (black bar), M. bovis BCG: ΔdosR (white bar), and complemented mutant (gray bar) was determined at each time point in the hypoxic shift down model. The experiment was carried out three times in triplicate, and results are given as means ± SD.

    Article Snippet: Bacterial Strains, Culture Media, and Chemicals M. tuberculosis H37Rv (ATCC 27294), M. bovis BCG Pasteur (ATCC 35734), derived mutant, and complemented strain, were maintained in Dubos broth (Difco) supplemented with 0.05% (v/v) Tween 80 and 10% Dubos Medium Albumin (Difco).

    Techniques: Mutagenesis

    Reconfirmation of hypoxic ATP IC 50 activity of HTS hits in hypoxic non-replicating mycobacteria. Ability to reduce the intracellular ATP levels of HTS hits was evaluated using various concentrations of compounds. A total of 866 hits showed activity against hypoxic M. bovis BCG MtbNar (a), and 283 hits showed activity against M. tuberculosis (b). The notation > 10 μM in panel a corresponds to compounds that showed at least 20% ATP reduction at 14.1 μM, and > 10 μM in panel b corresponds to compounds that showed at least 20% ATP reduction at 10 μM. The number of compounds belonging to each group and percentage of hits are depicted on the pieces in the pie chart.

    Journal: ACS Chemical Biology

    Article Title: A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis

    doi: 10.1021/cb2004884

    Figure Lengend Snippet: Reconfirmation of hypoxic ATP IC 50 activity of HTS hits in hypoxic non-replicating mycobacteria. Ability to reduce the intracellular ATP levels of HTS hits was evaluated using various concentrations of compounds. A total of 866 hits showed activity against hypoxic M. bovis BCG MtbNar (a), and 283 hits showed activity against M. tuberculosis (b). The notation > 10 μM in panel a corresponds to compounds that showed at least 20% ATP reduction at 14.1 μM, and > 10 μM in panel b corresponds to compounds that showed at least 20% ATP reduction at 10 μM. The number of compounds belonging to each group and percentage of hits are depicted on the pieces in the pie chart.

    Article Snippet: Bacterial Strains, Culture Media, and Chemicals M. tuberculosis H37Rv (ATCC 27294), M. bovis BCG Pasteur (ATCC 35734), derived mutant, and complemented strain, were maintained in Dubos broth (Difco) supplemented with 0.05% (v/v) Tween 80 and 10% Dubos Medium Albumin (Difco).

    Techniques: Activity Assay

    Esterase levels and activity correlate inversely with cellular TAG upon entry into and exit from non replicative-conditions. A and B , THL inhibits M. bovis BCG cultured in liquid ( A ) and solid media ( B ) with different potencies depending on the growth

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL) *

    doi: 10.1074/mcp.M113.029942

    Figure Lengend Snippet: Esterase levels and activity correlate inversely with cellular TAG upon entry into and exit from non replicative-conditions. A and B , THL inhibits M. bovis BCG cultured in liquid ( A ) and solid media ( B ) with different potencies depending on the growth

    Article Snippet: Mycobacterium bovis BCG Pasteur strain (ATCC 35734) and over-expression mutants were grown in Dubos liquid medium (Difco, USA) supplemented with 10% Dubos medium albumin (Difco), and 0.05% Tween 80, or in Middlebrook 7H11-OADC agar (Difco) at 37 °C.

    Techniques: Activity Assay, Cell Culture

    THL target spectrum in M. bovis BCG largely comprises lipid esterases. A , Schematic representation of the workflow and steps involved in identification of THL targets. M. bovis BCG was grown in three different physiological states in vitro , cells lysed

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL) *

    doi: 10.1074/mcp.M113.029942

    Figure Lengend Snippet: THL target spectrum in M. bovis BCG largely comprises lipid esterases. A , Schematic representation of the workflow and steps involved in identification of THL targets. M. bovis BCG was grown in three different physiological states in vitro , cells lysed

    Article Snippet: Mycobacterium bovis BCG Pasteur strain (ATCC 35734) and over-expression mutants were grown in Dubos liquid medium (Difco, USA) supplemented with 10% Dubos medium albumin (Difco), and 0.05% Tween 80, or in Middlebrook 7H11-OADC agar (Difco) at 37 °C.

    Techniques: In Vitro

    ClgR is a substrate of mycobacterial Clp, and its accumulation is toxic for M. bovis BCG. (A) Schematics of ClgR-RFP fusion proteins, their transformability, and colony color of M. bovis BCG transformants. Red, RFP; gray, ClgR. “Recovery” indicates whether transformants with the respective constructs could be obtained. “Color” indicates the color of the M. bovis BCG colonies. “GGSG” indicates the peptide sequence used as a short linker inserted between RFP and ClgR. Primers used and plasmid construction procedures employing the episomal plasmid pMV262 ( 18 ) to generate the respective strains are listed in Table S3 in the supplemental material. All ClgR-RFP fusion proteins as well as the ClgR nonfusion proteins were overexpressed in M. bovis BCG under the control of the constitutive P- hsp60 promoter ( 17 ). Note that the transformation efficiencies for the overexpression constructs for which colonies could be recovered were in the range of 5 × 10 4 /µg DNA and were similar for all constructs. Colony sizes were also similar with the exception of RFP-ClgR, ClgR-RFP-SsrA, and ClgR-RFP-ClgR(C9) transformants, which displayed somewhat smaller colony sizes. For overexpression constructs for which no transformants were obtained, the plates were incubated and observed for 2 months. (B) Fluorescence measurements of M. bovis BCG cultures carrying various ClgR-RFP fusion constructs shown in panel A without and with 24-h bortezomib treatment. (C) Effect of increasing bortezomib concentrations on fluorescence and growth of M. bovis BCG cultures expressing the RFP–full-length ClgR fusion protein (RFP-ClgR [A]). RFU, relative fluorescence units. The bacteria were grown in 96-well plates for 5 days as described in the text with a starting OD 600 of 0.05. Turbidity and fluorescence measurements were taken after day 5 with an Infinite M200 Pro plate reader (Tecan). Data shown in panels B and C represent means ± standard deviations from two biological and four technical replicates.

    Journal: mSphere

    Article Title: Mycobacterial Caseinolytic Protease Gene Regulator ClgR Is a Substrate of Caseinolytic Protease

    doi: 10.1128/mSphere.00338-16

    Figure Lengend Snippet: ClgR is a substrate of mycobacterial Clp, and its accumulation is toxic for M. bovis BCG. (A) Schematics of ClgR-RFP fusion proteins, their transformability, and colony color of M. bovis BCG transformants. Red, RFP; gray, ClgR. “Recovery” indicates whether transformants with the respective constructs could be obtained. “Color” indicates the color of the M. bovis BCG colonies. “GGSG” indicates the peptide sequence used as a short linker inserted between RFP and ClgR. Primers used and plasmid construction procedures employing the episomal plasmid pMV262 ( 18 ) to generate the respective strains are listed in Table S3 in the supplemental material. All ClgR-RFP fusion proteins as well as the ClgR nonfusion proteins were overexpressed in M. bovis BCG under the control of the constitutive P- hsp60 promoter ( 17 ). Note that the transformation efficiencies for the overexpression constructs for which colonies could be recovered were in the range of 5 × 10 4 /µg DNA and were similar for all constructs. Colony sizes were also similar with the exception of RFP-ClgR, ClgR-RFP-SsrA, and ClgR-RFP-ClgR(C9) transformants, which displayed somewhat smaller colony sizes. For overexpression constructs for which no transformants were obtained, the plates were incubated and observed for 2 months. (B) Fluorescence measurements of M. bovis BCG cultures carrying various ClgR-RFP fusion constructs shown in panel A without and with 24-h bortezomib treatment. (C) Effect of increasing bortezomib concentrations on fluorescence and growth of M. bovis BCG cultures expressing the RFP–full-length ClgR fusion protein (RFP-ClgR [A]). RFU, relative fluorescence units. The bacteria were grown in 96-well plates for 5 days as described in the text with a starting OD 600 of 0.05. Turbidity and fluorescence measurements were taken after day 5 with an Infinite M200 Pro plate reader (Tecan). Data shown in panels B and C represent means ± standard deviations from two biological and four technical replicates.

    Article Snippet: M. bovis BCG Pasteur ATCC 35734 (BCG) was purchased from the American Type Culture Collection and was grown at 37°C in Middlebrook 7H9 broth (BD Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, 0.0003% catalase, 0.2% glycerol, and 0.05% Tween 80.

    Techniques: Construct, Sequencing, Plasmid Preparation, Transformation Assay, Over Expression, Incubation, Fluorescence, Expressing

    Bortezomib treatment increases transcription of clpP1P2 , clgR , and acr2 genes in M. bovis BCG. (A) Bortezomib dose-dependent increase of RFP expression under the control of P- clpP1P2 , P- clgR , and P- acr2 promoters after 24 h of bortezomib treatment. RFU, relative fluorescence units. Primers and plasmid construction procedures using the integrative plasmid pMV306 ( 18 ) to generate the respective reporter strains are listed in Table S1 in the supplemental material. OD 600 was measured during the course of the experiment and was found to increase a maximum of 2-fold in the drug-free samples and less in the drug-containing samples. (B) Bortezomib-dependent increase of clpP1P2 , clgR , and acr2 mRNA. Transcript levels were measured after 16 h of bortezomib treatment. Primer sequences ( 16 , 19 ) can be found in Table S2 in the supplemental material. Relative expression (quantification cycle [ΔΔ C q ]) was calculated as described previously ( 20 ) by using 16S RNA as the reference. BZ, bortezomib. CIP, ciprofloxacin. MIC 90 , drug concentration that inhibited growth of the bacteria by 90%. MIC 90 of BZ, 12.5 µM. MIC 90 of CIP, 1.6 µM. Data in panels A and B are represented as means ± standard deviations from two biological and four technical replicates.

    Journal: mSphere

    Article Title: Mycobacterial Caseinolytic Protease Gene Regulator ClgR Is a Substrate of Caseinolytic Protease

    doi: 10.1128/mSphere.00338-16

    Figure Lengend Snippet: Bortezomib treatment increases transcription of clpP1P2 , clgR , and acr2 genes in M. bovis BCG. (A) Bortezomib dose-dependent increase of RFP expression under the control of P- clpP1P2 , P- clgR , and P- acr2 promoters after 24 h of bortezomib treatment. RFU, relative fluorescence units. Primers and plasmid construction procedures using the integrative plasmid pMV306 ( 18 ) to generate the respective reporter strains are listed in Table S1 in the supplemental material. OD 600 was measured during the course of the experiment and was found to increase a maximum of 2-fold in the drug-free samples and less in the drug-containing samples. (B) Bortezomib-dependent increase of clpP1P2 , clgR , and acr2 mRNA. Transcript levels were measured after 16 h of bortezomib treatment. Primer sequences ( 16 , 19 ) can be found in Table S2 in the supplemental material. Relative expression (quantification cycle [ΔΔ C q ]) was calculated as described previously ( 20 ) by using 16S RNA as the reference. BZ, bortezomib. CIP, ciprofloxacin. MIC 90 , drug concentration that inhibited growth of the bacteria by 90%. MIC 90 of BZ, 12.5 µM. MIC 90 of CIP, 1.6 µM. Data in panels A and B are represented as means ± standard deviations from two biological and four technical replicates.

    Article Snippet: M. bovis BCG Pasteur ATCC 35734 (BCG) was purchased from the American Type Culture Collection and was grown at 37°C in Middlebrook 7H9 broth (BD Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, 0.0003% catalase, 0.2% glycerol, and 0.05% Tween 80.

    Techniques: Expressing, Fluorescence, Plasmid Preparation, Concentration Assay

    Statins increase rifampin mycobactericidal effect. THP-1 cells were differentiated to macrophages with PMA, and after 24 h of rest, the cultures were treated with different regimes and were concomitantly infected with M. bovis (BCG Pasteur) (A), M. tuberculosis

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Statins Increase Rifampin Mycobactericidal Effect

    doi: 10.1128/AAC.01826-13

    Figure Lengend Snippet: Statins increase rifampin mycobactericidal effect. THP-1 cells were differentiated to macrophages with PMA, and after 24 h of rest, the cultures were treated with different regimes and were concomitantly infected with M. bovis (BCG Pasteur) (A), M. tuberculosis

    Article Snippet: M. bovis strain BCG Pasteur (ATCC 35734) and M. tuberculosis strain H37Rv were grown at 37°C in Middlebrook 7H9 base ADC enrichment medium (Becton Dickinson, Franklin Lakes, NJ), supplemented with 0.2% glycerol and 0.05% Tween 80 (Sigma).

    Techniques: Infection

    CD4 T cell proliferation and cytokine production induced by LPS- and BG-treated DC in a mixed lymphocyte reaction. M. bovis BCG-infected or uninfected BMDC were left untreated (UT) (open bars), stimulated with BG (MOI 1 and 5) (black bars) or stimulated with a normalized amount of LPS (23 or 113 EU/mL, respectively) (gray bars) in the presence of OTII OVA peptide for 24 h, before co-culture with transgenic T cells for 96 h. Division and proliferation indices of CD4 (A,B) T cells were derived. Proportion of naïve (CD62L + CD44 − ), activated (CD62L − CD44 + ) and double positive T cells were displayed as percentages of total CD4 T cells (C–E) . The levels of IFNγ, IL-4, and IL-12p40 produced after 96 h incubation were measured (F–H) . Results are expressed as mean ± SD of technical triplicates and are representative of two independent experiments. Significance values were derived using 1-way ANOVA with Holm-Sidak's multiple comparisons test (* p

    Journal: Frontiers in Immunology

    Article Title: Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity

    doi: 10.3389/fimmu.2019.02737

    Figure Lengend Snippet: CD4 T cell proliferation and cytokine production induced by LPS- and BG-treated DC in a mixed lymphocyte reaction. M. bovis BCG-infected or uninfected BMDC were left untreated (UT) (open bars), stimulated with BG (MOI 1 and 5) (black bars) or stimulated with a normalized amount of LPS (23 or 113 EU/mL, respectively) (gray bars) in the presence of OTII OVA peptide for 24 h, before co-culture with transgenic T cells for 96 h. Division and proliferation indices of CD4 (A,B) T cells were derived. Proportion of naïve (CD62L + CD44 − ), activated (CD62L − CD44 + ) and double positive T cells were displayed as percentages of total CD4 T cells (C–E) . The levels of IFNγ, IL-4, and IL-12p40 produced after 96 h incubation were measured (F–H) . Results are expressed as mean ± SD of technical triplicates and are representative of two independent experiments. Significance values were derived using 1-way ANOVA with Holm-Sidak's multiple comparisons test (* p

    Article Snippet: Bacteria, BG, and Mammalian Cell Culture Cultures of M. bovis BCG Pasteur strain (ATCC, 35734) were cultured in standing T25 flasks in Middlebrook 7H9 broth (Becton Dickinson Difco™, NJ, USA) at 37°C until OD600nm 0.5–0.7 and then stored at −80°C in 7H9 and 25% glycerol (1st Base, Singapore).

    Techniques: Infection, Co-Culture Assay, Transgenic Assay, Derivative Assay, Produced, Incubation

    Effect of BG treatment on lung bacterial loads in M. bovis BCG- and Mtb-infected mice. Adult female C57BL/6 mice were infected intratracheally (IT) with 10 6 CFU of M. bovis BCG (A,B,E) or 10 3 CFU Mtb H37Rv (C,D) . Treatment was administered as described in the legend of Figure 6 . For BG adjunct therapy, infected mice were treated daily with INH (0.05 mg/mL) or thrice weekly with Q203 (10 mg/kg) BDQ (15 mg/kg), DLM (10 mg/kg) or LZD (50 mg/kg) via the oral route, in combination with IT administration of BG as described in the legend of Figure 6 . Organs were harvested at day 35 p.i., unless otherwise indicated, and homogenates were plated for CFU enumeration. Data points are shown for each mouse ( n = 3–8 mice/group) and M. bovis BCG load (E) is expressed as a percentage of vehicle control. Significance values were derived using 1-way (E) or 2-way (A–D) ANOVA with Holm-Sidak's multiple comparisons test (* p

    Journal: Frontiers in Immunology

    Article Title: Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity

    doi: 10.3389/fimmu.2019.02737

    Figure Lengend Snippet: Effect of BG treatment on lung bacterial loads in M. bovis BCG- and Mtb-infected mice. Adult female C57BL/6 mice were infected intratracheally (IT) with 10 6 CFU of M. bovis BCG (A,B,E) or 10 3 CFU Mtb H37Rv (C,D) . Treatment was administered as described in the legend of Figure 6 . For BG adjunct therapy, infected mice were treated daily with INH (0.05 mg/mL) or thrice weekly with Q203 (10 mg/kg) BDQ (15 mg/kg), DLM (10 mg/kg) or LZD (50 mg/kg) via the oral route, in combination with IT administration of BG as described in the legend of Figure 6 . Organs were harvested at day 35 p.i., unless otherwise indicated, and homogenates were plated for CFU enumeration. Data points are shown for each mouse ( n = 3–8 mice/group) and M. bovis BCG load (E) is expressed as a percentage of vehicle control. Significance values were derived using 1-way (E) or 2-way (A–D) ANOVA with Holm-Sidak's multiple comparisons test (* p

    Article Snippet: Bacteria, BG, and Mammalian Cell Culture Cultures of M. bovis BCG Pasteur strain (ATCC, 35734) were cultured in standing T25 flasks in Middlebrook 7H9 broth (Becton Dickinson Difco™, NJ, USA) at 37°C until OD600nm 0.5–0.7 and then stored at −80°C in 7H9 and 25% glycerol (1st Base, Singapore).

    Techniques: Infection, Mouse Assay, Derivative Assay

    Activation status and cytokine production of WT vs. ΔTLR4 BMMO and BMDC upon BG and LPS treatment. Uninfected and M. bovis BCG-infected BMMO and BMDC were treated with BG at MOI 40 and 5, respectively, or with a normalized amount of LPS (1,500 and 188 EU/mL, respectively) or left untreated (UT) for 24 h before analysis of surface activation markers by flow cytometry. Expression levels of CD80, CD86, CD54 for BMMO (A–C) and CD86, CD40, and CCR7 for BMDC (D–F) are shown for WT (open bar) and ΔTLR4 (black bar) cells. Cell culture supernatants were collected to quantify the levels of TNFα, IL-6, IL-10, IL12p40, and IL-12p70 produced by ELISA. Cytokine production was measured in WT and ΔTLR4 BMMO (G–J) and BMDC (K–O) . Results are expressed as mean ± SD of technical triplicates (A–F) or duplicates (G–O) and are representative of two independent experiments. Significance values were derived using 2-way ANOVA with Holm-Sidak's multiple comparisons test (ns: not significant, * p

    Journal: Frontiers in Immunology

    Article Title: Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity

    doi: 10.3389/fimmu.2019.02737

    Figure Lengend Snippet: Activation status and cytokine production of WT vs. ΔTLR4 BMMO and BMDC upon BG and LPS treatment. Uninfected and M. bovis BCG-infected BMMO and BMDC were treated with BG at MOI 40 and 5, respectively, or with a normalized amount of LPS (1,500 and 188 EU/mL, respectively) or left untreated (UT) for 24 h before analysis of surface activation markers by flow cytometry. Expression levels of CD80, CD86, CD54 for BMMO (A–C) and CD86, CD40, and CCR7 for BMDC (D–F) are shown for WT (open bar) and ΔTLR4 (black bar) cells. Cell culture supernatants were collected to quantify the levels of TNFα, IL-6, IL-10, IL12p40, and IL-12p70 produced by ELISA. Cytokine production was measured in WT and ΔTLR4 BMMO (G–J) and BMDC (K–O) . Results are expressed as mean ± SD of technical triplicates (A–F) or duplicates (G–O) and are representative of two independent experiments. Significance values were derived using 2-way ANOVA with Holm-Sidak's multiple comparisons test (ns: not significant, * p

    Article Snippet: Bacteria, BG, and Mammalian Cell Culture Cultures of M. bovis BCG Pasteur strain (ATCC, 35734) were cultured in standing T25 flasks in Middlebrook 7H9 broth (Becton Dickinson Difco™, NJ, USA) at 37°C until OD600nm 0.5–0.7 and then stored at −80°C in 7H9 and 25% glycerol (1st Base, Singapore).

    Techniques: Activation Assay, Infection, Flow Cytometry, Cytometry, Expressing, Cell Culture, Produced, Enzyme-linked Immunosorbent Assay, Derivative Assay

    NO production in BMMO upon BG and LPS treatment and killing efficacy. (A) BMMO were left untreated (UT), or treated with 10 ng/mL LPS, or with a combination of (20 ng/mL IFNγ +10 ng/mL LPS), or with a range of BG MOIs for 24 h before nitrite determination. (B) Uninfected or M. bovis BCG-infected WT and ΔTLR4 BMMO were treated with BG at MOI 40 (BG40) or with a normalized amount of LPS (LPS40, corresponding to 1,500 EU/mL) or left untreated (UT) for 24 h before nitrite quantification. (C) M. bovis BCG-infected BMMO were treated with BG (MOI 40) or left untreated (UT). Intracellular bacterial loads were determined at 1, 3, 5, and 7 days post-infection. Results are expressed as mean ± SD of technical triplicates and are representative of two independent experiments. Significance values were derived using 1-way ANOVA for (A) and 2-way ANOVA for (B,C) with Holm-Sidak's multiple comparisons test (** p

    Journal: Frontiers in Immunology

    Article Title: Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity

    doi: 10.3389/fimmu.2019.02737

    Figure Lengend Snippet: NO production in BMMO upon BG and LPS treatment and killing efficacy. (A) BMMO were left untreated (UT), or treated with 10 ng/mL LPS, or with a combination of (20 ng/mL IFNγ +10 ng/mL LPS), or with a range of BG MOIs for 24 h before nitrite determination. (B) Uninfected or M. bovis BCG-infected WT and ΔTLR4 BMMO were treated with BG at MOI 40 (BG40) or with a normalized amount of LPS (LPS40, corresponding to 1,500 EU/mL) or left untreated (UT) for 24 h before nitrite quantification. (C) M. bovis BCG-infected BMMO were treated with BG (MOI 40) or left untreated (UT). Intracellular bacterial loads were determined at 1, 3, 5, and 7 days post-infection. Results are expressed as mean ± SD of technical triplicates and are representative of two independent experiments. Significance values were derived using 1-way ANOVA for (A) and 2-way ANOVA for (B,C) with Holm-Sidak's multiple comparisons test (** p

    Article Snippet: Bacteria, BG, and Mammalian Cell Culture Cultures of M. bovis BCG Pasteur strain (ATCC, 35734) were cultured in standing T25 flasks in Middlebrook 7H9 broth (Becton Dickinson Difco™, NJ, USA) at 37°C until OD600nm 0.5–0.7 and then stored at −80°C in 7H9 and 25% glycerol (1st Base, Singapore).

    Techniques: Infection, Derivative Assay

    Immune cell populations and cytokine production in lungs from BG-treated vs. LPS-treated M. bovis BCG-infected mice. Adult female C57BL/6 mice were infected intratracheally (IT) with 10 6 CFU of M. bovis BCG. Starting from day 7 post-infection, mice were administered IT weekly and for 4 consecutive weeks with 10 6 BG, a normalized amount of LPS (75 EU), vehicle or were left untreated (UT). Lungs were harvested 1 day (day 29 p.i.) or 1 week (day 35 p.i.) after the last dose administered and the myeloid (A–F) and CD4 T cell (G–K) populations were analyzed by flow cytometry. Levels of TNFα, IFNγ, IL-6, IL-10, IL12p40, and IL-12p70 in the lung homogenates (L–O) were determined by ELISA. Results are representative of two independent experiments and data points are shown for each mouse ( n = 4–5 mice/group). Significance values were derived using 2-way ANOVA with Holm-Sidak's multiple comparisons test (* p

    Journal: Frontiers in Immunology

    Article Title: Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity

    doi: 10.3389/fimmu.2019.02737

    Figure Lengend Snippet: Immune cell populations and cytokine production in lungs from BG-treated vs. LPS-treated M. bovis BCG-infected mice. Adult female C57BL/6 mice were infected intratracheally (IT) with 10 6 CFU of M. bovis BCG. Starting from day 7 post-infection, mice were administered IT weekly and for 4 consecutive weeks with 10 6 BG, a normalized amount of LPS (75 EU), vehicle or were left untreated (UT). Lungs were harvested 1 day (day 29 p.i.) or 1 week (day 35 p.i.) after the last dose administered and the myeloid (A–F) and CD4 T cell (G–K) populations were analyzed by flow cytometry. Levels of TNFα, IFNγ, IL-6, IL-10, IL12p40, and IL-12p70 in the lung homogenates (L–O) were determined by ELISA. Results are representative of two independent experiments and data points are shown for each mouse ( n = 4–5 mice/group). Significance values were derived using 2-way ANOVA with Holm-Sidak's multiple comparisons test (* p

    Article Snippet: Bacteria, BG, and Mammalian Cell Culture Cultures of M. bovis BCG Pasteur strain (ATCC, 35734) were cultured in standing T25 flasks in Middlebrook 7H9 broth (Becton Dickinson Difco™, NJ, USA) at 37°C until OD600nm 0.5–0.7 and then stored at −80°C in 7H9 and 25% glycerol (1st Base, Singapore).

    Techniques: Infection, Mouse Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay

    Representative high resolution melting graphs corresponding to one high resolution melting analysis of a subset of cultured samples (n = 22). Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis/M. bovis BCG in red and M. caprae in green. ( a ) Melting curves; ( b ) Normalized plot; ( c ) Difference plot.

    Journal: Scientific Reports

    Article Title: Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples

    doi: 10.1038/s41598-018-38243-6

    Figure Lengend Snippet: Representative high resolution melting graphs corresponding to one high resolution melting analysis of a subset of cultured samples (n = 22). Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis/M. bovis BCG in red and M. caprae in green. ( a ) Melting curves; ( b ) Normalized plot; ( c ) Difference plot.

    Article Snippet: In order to generate difference plots, normalized fluorescence data of sample curves were subtracted from a reference curve of M. bovis BCG Pasteur ATCC 35734 to visually accentuate differences in a greater resolution.

    Techniques: Cell Culture

    Representative high resolution melting graphs corresponding to one high resolution melting analysis of a subset of clinical specimens (n = 19). Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis/M. bovis BCG in red and M. caprae in green. ( a ) Melting curves; ( b ) Normalized plot; ( c ) Difference plot.

    Journal: Scientific Reports

    Article Title: Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples

    doi: 10.1038/s41598-018-38243-6

    Figure Lengend Snippet: Representative high resolution melting graphs corresponding to one high resolution melting analysis of a subset of clinical specimens (n = 19). Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis/M. bovis BCG in red and M. caprae in green. ( a ) Melting curves; ( b ) Normalized plot; ( c ) Difference plot.

    Article Snippet: In order to generate difference plots, normalized fluorescence data of sample curves were subtracted from a reference curve of M. bovis BCG Pasteur ATCC 35734 to visually accentuate differences in a greater resolution.

    Techniques:

    TTD of M. bovis BCG samples. Plot of bulk capacitance obtained over time for samples with a low initial loads (~1 × 10 3 CFU/ml) and b high initial loads (~1 × 10 5 CFU/ml) for M. bovis BCG . The error bars indicate the standard deviation of the readings (n = 5) taken at each time interval. Statistical analysis is used to compare the baseline reading with the various time interval readings (U critical = 2). [ NSH not significantly higher, SH significantly higher]

    Journal: Biological Research

    Article Title: Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

    doi: 10.1186/s40659-017-0126-7

    Figure Lengend Snippet: TTD of M. bovis BCG samples. Plot of bulk capacitance obtained over time for samples with a low initial loads (~1 × 10 3 CFU/ml) and b high initial loads (~1 × 10 5 CFU/ml) for M. bovis BCG . The error bars indicate the standard deviation of the readings (n = 5) taken at each time interval. Statistical analysis is used to compare the baseline reading with the various time interval readings (U critical = 2). [ NSH not significantly higher, SH significantly higher]

    Article Snippet: Mycobacterial cell culture Slow growing acid-fast organism M. bovis BCG (ATCC® 35734™) and rapid growing mycobacteria M. smegmatis (ATCC 700084) are used.

    Techniques: Standard Deviation

    Bioxy vs. Mycobacterium bovis

    Journal: PLoS ONE

    Article Title: The wide spectrum high biocidal potency of Bioxy formulation when dissolved in water at different concentrations

    doi: 10.1371/journal.pone.0172224

    Figure Lengend Snippet: Bioxy vs. Mycobacterium bovis

    Article Snippet: Bioxy vs. Mycobacterium bovis (bovine tuberculosis) Mycobacterium bovis strain ATCC 35743 was prepared from a frozen suspension grown in modified Proskauer-Beck medium [asparagine (Difco), magnesium sulphate (JT Baker), magnesium citrate (Sigma-Aldrich), and glycerol (Fisher)].

    Techniques:

    Bioxy vs. Mycobacterium bovis

    Journal: PLoS ONE

    Article Title: The wide spectrum high biocidal potency of Bioxy formulation when dissolved in water at different concentrations

    doi: 10.1371/journal.pone.0172224

    Figure Lengend Snippet: Bioxy vs. Mycobacterium bovis

    Article Snippet: Bioxy vs. Mycobacterium bovis (bovine tuberculosis) Mycobacterium bovis strain ATCC 35743 was prepared from a frozen suspension grown in modified Proskauer-Beck medium [asparagine (Difco), magnesium sulphate (JT Baker), magnesium citrate (Sigma-Aldrich), and glycerol (Fisher)].

    Techniques:

    DosR Asp-54 is required for gene induction but not for binding. EMSA of mutant DosR (Asp-54Glu) with labelled acr promoter DNA. Lanes: 1, no extract; 2, induced extract of DosR (Asp-54Glu); 3, induced extract of DosR (WT). Hypoxic induction of acr by mutant DosR (Asp-54Glu). Acr expression measured by luciferase reporter gene in BCG and in BCG with DosR (Asp-54Glu) replacing DosR (WT). Shown are representative data from one of two experiments, each of which was performed in duplicate.

    Journal: Molecular microbiology

    Article Title: Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis

    doi:

    Figure Lengend Snippet: DosR Asp-54 is required for gene induction but not for binding. EMSA of mutant DosR (Asp-54Glu) with labelled acr promoter DNA. Lanes: 1, no extract; 2, induced extract of DosR (Asp-54Glu); 3, induced extract of DosR (WT). Hypoxic induction of acr by mutant DosR (Asp-54Glu). Acr expression measured by luciferase reporter gene in BCG and in BCG with DosR (Asp-54Glu) replacing DosR (WT). Shown are representative data from one of two experiments, each of which was performed in duplicate.

    Article Snippet: H37Rv (ATCC 27294), H37Rv:Δ dosR::kan and M. bovis BCG-Montreal (ATCC 35735) were grown to mid-log phase in 7H9 media with 0.05% Tween 80 and ADC supplement (Becton Dickinson) and stored as 1 ml aliquots in 15% glycerol (final concentration) at −80°C.

    Techniques: Binding Assay, Mutagenesis, Expressing, Luciferase

    Analysis of trans -dominant mutants by gel electrophoresis. (a) Peroxidase activity gel of katG mutant plasmids in M. bovis BCG strain ATCC 35735 (lanes 1 to 8). Lanes 9 and 10 show vector control (VC) and the R463L mutant plasmid in the Δ katG strain ATCC 35747. (b) Nondenaturing gel transferred to PVDF membrane and probed with anti-KatG polyclonal antibody. Lane assignments are identical to those for panel a. (c) Denaturing gel transferred to PVDF membrane and probed with anti-KatG polyclonal antibody. Lane assignments are identical to those in panel a.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Exploring the Structure and Function of the Mycobacterial KatG Protein Using trans-Dominant Mutants

    doi: 10.1128/AAC.47.1.188-195.2003

    Figure Lengend Snippet: Analysis of trans -dominant mutants by gel electrophoresis. (a) Peroxidase activity gel of katG mutant plasmids in M. bovis BCG strain ATCC 35735 (lanes 1 to 8). Lanes 9 and 10 show vector control (VC) and the R463L mutant plasmid in the Δ katG strain ATCC 35747. (b) Nondenaturing gel transferred to PVDF membrane and probed with anti-KatG polyclonal antibody. Lane assignments are identical to those for panel a. (c) Denaturing gel transferred to PVDF membrane and probed with anti-KatG polyclonal antibody. Lane assignments are identical to those in panel a.

    Article Snippet: In order to probe the structure and function of the M. tuberculosis KatG protein, various point mutants of the enzyme were episomally expressed from their native promoter in the attenuated strain of M. bovis (BCG) ATCC 35735.

    Techniques: Nucleic Acid Electrophoresis, Activity Assay, Mutagenesis, Plasmid Preparation

    Analysis of katG point mutants by native gel electrophoresis. (a) Peroxidase activity gel of katG mutant plasmids in the Δ katG strain ATCC 35747 (lanes 1 to 7). Lane 8 shows vector control (VC) in the katG + M. bovis (BCG) strain ATCC 35735. (b) Nondenaturing gel transferred to PVDF membrane and probed with anti-KatG polyclonal antibody. Lane assignments are identical to those in panel a. Although barely visible, the pD381G mutant does show a protein band with altered mobility (similar to the pT275P mutant).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Exploring the Structure and Function of the Mycobacterial KatG Protein Using trans-Dominant Mutants

    doi: 10.1128/AAC.47.1.188-195.2003

    Figure Lengend Snippet: Analysis of katG point mutants by native gel electrophoresis. (a) Peroxidase activity gel of katG mutant plasmids in the Δ katG strain ATCC 35747 (lanes 1 to 7). Lane 8 shows vector control (VC) in the katG + M. bovis (BCG) strain ATCC 35735. (b) Nondenaturing gel transferred to PVDF membrane and probed with anti-KatG polyclonal antibody. Lane assignments are identical to those in panel a. Although barely visible, the pD381G mutant does show a protein band with altered mobility (similar to the pT275P mutant).

    Article Snippet: In order to probe the structure and function of the M. tuberculosis KatG protein, various point mutants of the enzyme were episomally expressed from their native promoter in the attenuated strain of M. bovis (BCG) ATCC 35735.

    Techniques: Nucleic Acid Electrophoresis, Activity Assay, Mutagenesis, Plasmid Preparation

    Linear regression curves, R 2 values, and confidence and prediction intervals (95%) corresponding to devR , IS 1311 , and ITSdenak probe C T values in spiked samples with different concentrations of M. bovis BCG (A), M. avium subsp. hominissuis (B), or M.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

    doi: 10.1128/JCM.03168-14

    Figure Lengend Snippet: Linear regression curves, R 2 values, and confidence and prediction intervals (95%) corresponding to devR , IS 1311 , and ITSdenak probe C T values in spiked samples with different concentrations of M. bovis BCG (A), M. avium subsp. hominissuis (B), or M.

    Article Snippet: The mycobacterial strains used for inoculation experiments included Mycobacterium bovis BCG CCUG 27863, M. avium subsp. hominissuis strain 104, and M. kansasii ATCC 12478.

    Techniques:

    Neutralization of interleukin-27 (IL-27) improves neonatal innate and adaptive immune responses. (a) Cord blood macrophages were treated with sIL-27R or medium alone for 4 hr and then infected with bacillus Calmette–Guérin (BCG; multiplicity

    Journal: Immunology

    Article Title: Neonatal macrophages express elevated levels of interleukin-27 that oppose immune responses

    doi: 10.1111/imm.12095

    Figure Lengend Snippet: Neutralization of interleukin-27 (IL-27) improves neonatal innate and adaptive immune responses. (a) Cord blood macrophages were treated with sIL-27R or medium alone for 4 hr and then infected with bacillus Calmette–Guérin (BCG; multiplicity

    Article Snippet: Mycobacterium bovis bacillus Calmette–Guérin (BCG) was purchased from the American Type Culture Collection (ATCC, Manassas, VA).

    Techniques: Neutralization, Infection

    (a) Nucleosomal-fragmentation ELISA of THP-1 cells uninfected (Un) or infected with avirulent BCG or virulent wild-type M. bovis . Data are means plus standard errors of three experiments. ∗, statistically significant difference between groups ( P

    Journal: Infection and Immunity

    Article Title: THP-1 Cell Apoptosis in Response to Mycobacterial Infection

    doi: 10.1128/IAI.71.1.254-259.2003

    Figure Lengend Snippet: (a) Nucleosomal-fragmentation ELISA of THP-1 cells uninfected (Un) or infected with avirulent BCG or virulent wild-type M. bovis . Data are means plus standard errors of three experiments. ∗, statistically significant difference between groups ( P

    Article Snippet: Cultures of wild-type Mycobacterium bovis , M. bovis bacillus Calmette-Guérin (BCG), M. tuberculosis H37Ra, and H37Rv were obtained from the American Type Culture Collection.

    Techniques: Enzyme-linked Immunosorbent Assay, Infection

    Colorimetric caspase activity assay 12 h postinfection shows an increase in protease activity of caspases 8, 9, and 10 due to BCG (solid bars) but not to M. bovis (hatched bars).

    Journal: Infection and Immunity

    Article Title: THP-1 Cell Apoptosis in Response to Mycobacterial Infection

    doi: 10.1128/IAI.71.1.254-259.2003

    Figure Lengend Snippet: Colorimetric caspase activity assay 12 h postinfection shows an increase in protease activity of caspases 8, 9, and 10 due to BCG (solid bars) but not to M. bovis (hatched bars).

    Article Snippet: Cultures of wild-type Mycobacterium bovis , M. bovis bacillus Calmette-Guérin (BCG), M. tuberculosis H37Ra, and H37Rv were obtained from the American Type Culture Collection.

    Techniques: Caspase Activity Assay, Activity Assay

    Identification of the binding sites for OxyS in the promoter region of katG . ( A ) DNase I footprinting assays were employed to assess the binding sequence of OxyS. The experiments were carried out as described under the “ Materials and Methods ” section. The ladders are shown in the right panel and the nucleotide sequences obtained are listed. The protected regions are underlined. ( B ) Summary of OxyS footprinting analysis in the M. tuberculosis katG promoter region. The DNA sequence where OxyS was found to bind corresponds with the katG promoter region from −180 to −1. OxyS footprint regions (underlined) and the position of the katG translation start site are indicated. The T-N 11 -A motif conserved in the DNA binding sites of LysR regulators is indicated on the sequence. Two OxyS binding boxes are indicated. ( C ) A blast assay for the conserved sequence motif recognition by OxyS (OxyS box1) among different mycobacterial species. Sequence alignment was carried and visualized locally using the BioEdit software. The conserved T-N 11 -A DNA-binding site in LysR regulators is shown at the bottom. M. tu, Mycobacterium tuberculosis H37Rv; M. bo, Mycobacterium bovis AF2122/97; M. in, Mycobacterium intracellulare ATCC 13950; M. av, Mycobacterium avium subsp. paratuberculosis K-10; M. sm, Mycobacterium smegmatis str. mc 2 155; M. ka, Mycobacterium kansasii ATCC 12478; M. ma, Mycobacterium marinum M; M. ul, Mycobacterium ulcerans Agy99. ( D ) DNA-binding assays for OxyS on OxyS box1 and OxyS box1mut substrates. The EMSA reactions (10 µl) for measuring mobility shift contained FITC-labeled DNA substrate and increasing amount of OxyS (0 nM, 100 nM, 200 nM and 300 nM). The conserved “TG” and “GA” in OxyS box1 were replaced by “CC” in OxyS box1mut substrate. The protein/DNA complexes are indicated by arrows on the right of the panels.

    Journal: PLoS ONE

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene

    doi: 10.1371/journal.pone.0030186

    Figure Lengend Snippet: Identification of the binding sites for OxyS in the promoter region of katG . ( A ) DNase I footprinting assays were employed to assess the binding sequence of OxyS. The experiments were carried out as described under the “ Materials and Methods ” section. The ladders are shown in the right panel and the nucleotide sequences obtained are listed. The protected regions are underlined. ( B ) Summary of OxyS footprinting analysis in the M. tuberculosis katG promoter region. The DNA sequence where OxyS was found to bind corresponds with the katG promoter region from −180 to −1. OxyS footprint regions (underlined) and the position of the katG translation start site are indicated. The T-N 11 -A motif conserved in the DNA binding sites of LysR regulators is indicated on the sequence. Two OxyS binding boxes are indicated. ( C ) A blast assay for the conserved sequence motif recognition by OxyS (OxyS box1) among different mycobacterial species. Sequence alignment was carried and visualized locally using the BioEdit software. The conserved T-N 11 -A DNA-binding site in LysR regulators is shown at the bottom. M. tu, Mycobacterium tuberculosis H37Rv; M. bo, Mycobacterium bovis AF2122/97; M. in, Mycobacterium intracellulare ATCC 13950; M. av, Mycobacterium avium subsp. paratuberculosis K-10; M. sm, Mycobacterium smegmatis str. mc 2 155; M. ka, Mycobacterium kansasii ATCC 12478; M. ma, Mycobacterium marinum M; M. ul, Mycobacterium ulcerans Agy99. ( D ) DNA-binding assays for OxyS on OxyS box1 and OxyS box1mut substrates. The EMSA reactions (10 µl) for measuring mobility shift contained FITC-labeled DNA substrate and increasing amount of OxyS (0 nM, 100 nM, 200 nM and 300 nM). The conserved “TG” and “GA” in OxyS box1 were replaced by “CC” in OxyS box1mut substrate. The protein/DNA complexes are indicated by arrows on the right of the panels.

    Article Snippet: M. tu, Mycobacterium tuberculosis H37Rv; M. bo, Mycobacterium bovis AF2122/97; M. ka, Mycobacterium kansasii ATCC 12478; M. ma, Mycobacterium marinum M; M. le, Mycobacterium leprae TN; M. av, Mycobacterium avium subsp. paratuberculosis K-10; M. in, Mycobacterium intracellulare ATCC 13950; M. ul, Mycobacterium ulcerans Agy99; M. sm, Mycobacterium smegmatis str. mc2 155. (TIF) Click here for additional data file.

    Techniques: Binding Assay, Footprinting, Sequencing, Software, Mobility Shift, Labeling

    Southern blot of chromosomal DNAs from 15 mycobacterial species. DNA was digested with Not I and probed with the sodC gene from M. tuberculosis . Lane 1, M. tuberculosis; lane 2, M. africanum; lane 3, M. bovis; lane 4, M. microti; lane 5, M. bovis BCG; lane 6, M. fortuitum; lane 7, M. chelonae; lane 8, M. smegmatis ; lane 9, M. avium; lane 10, M. intracellulare; lane 11, M. gordonii; lane 12, M. marinum; lane 13, M. scrofulaceum ; lane 14, M. kansasii; and lane 15, M. xenopi .

    Journal: Infection and Immunity

    Article Title: Cu,Zn Superoxide Dismutase of Mycobacterium tuberculosis Contributes to Survival in Activated Macrophages That Are Generating an Oxidative Burst

    doi: 10.1128/IAI.69.8.4980-4987.2001

    Figure Lengend Snippet: Southern blot of chromosomal DNAs from 15 mycobacterial species. DNA was digested with Not I and probed with the sodC gene from M. tuberculosis . Lane 1, M. tuberculosis; lane 2, M. africanum; lane 3, M. bovis; lane 4, M. microti; lane 5, M. bovis BCG; lane 6, M. fortuitum; lane 7, M. chelonae; lane 8, M. smegmatis ; lane 9, M. avium; lane 10, M. intracellulare; lane 11, M. gordonii; lane 12, M. marinum; lane 13, M. scrofulaceum ; lane 14, M. kansasii; and lane 15, M. xenopi .

    Article Snippet: The bacterial strains used in this study included M. tuberculosis strain Erdman (ATCC 35801), Mycobacterium bovis El Paso, Mycobacterium africanum , Mycobacterium microti , M. bovis BCG Pasteur, Mycobacterium gordonii, Mycobacterium xenopi, Mycobacterium smegmatis (ATCC 607), Mycobacterium chelonae subsp. chelonae (ATCC 35752), Mycobacterium fortuitum subsp. peregrinum (ATCC 14467), Mycobacterium kansasii (clinical isolate), Mycobacterium scrofulaceum (clinical isolate), Mycobacterium marinum (ATC 927), Mycobacterium avium (clinical isolate), and Mycobacterium intracellulare (clinical isolate).

    Techniques: Southern Blot

    In vivo expression of M. tuberculosis response regulators in macrophages. Response regulator promoter fusions were transformed into M. tuberculosis H37Rv or M. bovis bacillus Calmette–Guérin Pasteur and analyzed for expression during growth in macrophages by epifluorescence microscopy. Semiquantitative PCR was used to analyze differences in reporter plasmid copy number between strains. M. tuberculosis response regulator fusions not shown exhibited no detectable fluorescence in M. bovis bacillus Calmette–Guérin or in M. tuberculosis during growth in vitro or during growth in macrophages. +, 0–100 relative fluorescence units (RFU); ++, 100–225 RFU; +++, 225–450 RFU.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mycobacterium tuberculosis signal transduction system required for persistent infections

    doi: 10.1073/pnas.221272198

    Figure Lengend Snippet: In vivo expression of M. tuberculosis response regulators in macrophages. Response regulator promoter fusions were transformed into M. tuberculosis H37Rv or M. bovis bacillus Calmette–Guérin Pasteur and analyzed for expression during growth in macrophages by epifluorescence microscopy. Semiquantitative PCR was used to analyze differences in reporter plasmid copy number between strains. M. tuberculosis response regulator fusions not shown exhibited no detectable fluorescence in M. bovis bacillus Calmette–Guérin or in M. tuberculosis during growth in vitro or during growth in macrophages. +, 0–100 relative fluorescence units (RFU); ++, 100–225 RFU; +++, 225–450 RFU.

    Article Snippet: M. tuberculosis H37Rv (ATCC 27294), Mycobacterium bovis bacillus Calmette–Guérin Pasteur (ATCC 27291), and Escherichia coli DH5α were used.

    Techniques: In Vivo, Expressing, Transformation Assay, Epifluorescence Microscopy, Polymerase Chain Reaction, Plasmid Preparation, Fluorescence, In Vitro

    Epifluorescence microscopy of macrophage-grown M. tuberculosis H37Rv or M. bovis bacillus Calmette–Guérin representative response regulator promoter fusions from class I ( A – C ), class III ( D – F ), class IV ( G – I ), or the hsp60-gfp control ( J – L ) are shown. Mean fluorescence intensity was determined from M. bovis bacillus Calmette–Guérin-infected J774 monolayers ( A , D , G , and J ), M. tuberculosis H37Rv-infected J774 monolayers ( B , E , H , and K ), or M. tuberculosis H37Rv-infected human peripheral blood monocyte-derived macrophages ( C , F , I , and L ). Insets show intracellular bacilli stained with rhodamine-auramine.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mycobacterium tuberculosis signal transduction system required for persistent infections

    doi: 10.1073/pnas.221272198

    Figure Lengend Snippet: Epifluorescence microscopy of macrophage-grown M. tuberculosis H37Rv or M. bovis bacillus Calmette–Guérin representative response regulator promoter fusions from class I ( A – C ), class III ( D – F ), class IV ( G – I ), or the hsp60-gfp control ( J – L ) are shown. Mean fluorescence intensity was determined from M. bovis bacillus Calmette–Guérin-infected J774 monolayers ( A , D , G , and J ), M. tuberculosis H37Rv-infected J774 monolayers ( B , E , H , and K ), or M. tuberculosis H37Rv-infected human peripheral blood monocyte-derived macrophages ( C , F , I , and L ). Insets show intracellular bacilli stained with rhodamine-auramine.

    Article Snippet: M. tuberculosis H37Rv (ATCC 27294), Mycobacterium bovis bacillus Calmette–Guérin Pasteur (ATCC 27291), and Escherichia coli DH5α were used.

    Techniques: Epifluorescence Microscopy, Fluorescence, Infection, Derivative Assay, Staining

    BCG and M. brumae survival inside T24, MB49 and 5637 BC cells. Colony-forming units (CFUs) from cell lysates at different time-points after infection are represented (h, hours). Values represent the means ± SEMs of three serial dilutions of triplicate culture wells of M. brumae infected T24 cells ( a ), MB49 cells ( b ) and 5637 cells ( c ), and BCG-infected T24 cells ( a ). Data are representative of one out of three (for T24) and two (for MB49 and 5637) independent experiments. BCG counts were estimated from the results obtained in the 96-well plate 7H10-based spot assay from dilutions of infected MB49 and 5637 BC cells.

    Journal: Scientific Reports

    Article Title: Mycobacteria emulsified in olive oil-in-water trigger a robust immune response in bladder cancer treatment

    doi: 10.1038/srep27232

    Figure Lengend Snippet: BCG and M. brumae survival inside T24, MB49 and 5637 BC cells. Colony-forming units (CFUs) from cell lysates at different time-points after infection are represented (h, hours). Values represent the means ± SEMs of three serial dilutions of triplicate culture wells of M. brumae infected T24 cells ( a ), MB49 cells ( b ) and 5637 cells ( c ), and BCG-infected T24 cells ( a ). Data are representative of one out of three (for T24) and two (for MB49 and 5637) independent experiments. BCG counts were estimated from the results obtained in the 96-well plate 7H10-based spot assay from dilutions of infected MB49 and 5637 BC cells.

    Article Snippet: Bacterial strains and cell lines M. bovis BCG Connaught (ATCC 35745) and M. brumae (ATCC 51384) were grown on Middlebrook 7H10 agar (Difco Laboratories, Surrey, UK) supplemented with 10% oleic-albumin-dextrose-catalase enrichment medium at 37 °C for 4 and 1 weeks, respectively.

    Techniques: Infection, Spot Test

    M. bovis BCG infection and a 19-kDa mycobacterial lipoprotein inhibit IFN-γ-inducible MHC-II expression on alveolar macrophages. (A to D) Alveolar macrophages were cultured with or without BCG at an MOI of 3 to 5 and with or without the 19-kDa lipoprotein (250 ng/ml) for 24 h. IFN-γ (2 ng/ml) was added after infection or pretreatment with the 19-kDa lipoprotein, and alveolar macrophages were cultured for an additional 24, 48, and 72 h in the presence of IFN-γ. The expression of MHC-II was measured by flow cytometry, and histograms representative of four independent experiments are shown. IFN-γ-induced MHC-II expression on BCG-infected or 19-kDa-lipoprotein-treated macrophages (black lines) is compared to control treated cells (shaded) after 48 and 72 h of IFN-γ exposure. Isotype control staining for both treatment groups is shown with overlapping thin lines. (E to F) Alveolar macrophages were isolated, cultured overnight, and then treated without (medium) or with the 19-kDa lipoprotein (500 ng/ml) for 24 h. After 24 h, fresh 19-kDa lipoprotein was added along with IFN-γ (2 ng/ml), and cells were incubated for an additional 48 h. CIITA and MHC-II mRNA expression was measured by real-time PCR and normalized to GAPDH expression. Data from a representative experiment ( n = 3) are shown. Decreases in CIITA and MHC-II mRNA expression were analyzed by using a paired Student's t test, and significant differences are designated with asterisks ( P ≤ 0.05).

    Journal: Infection and Immunity

    Article Title: Inhibition of Major Histocompatibility Complex II Expression and Antigen Processing in Murine Alveolar Macrophages by Mycobacterium bovis BCG and the 19-Kilodalton Mycobacterial Lipoprotein

    doi: 10.1128/IAI.72.4.2101-2110.2004

    Figure Lengend Snippet: M. bovis BCG infection and a 19-kDa mycobacterial lipoprotein inhibit IFN-γ-inducible MHC-II expression on alveolar macrophages. (A to D) Alveolar macrophages were cultured with or without BCG at an MOI of 3 to 5 and with or without the 19-kDa lipoprotein (250 ng/ml) for 24 h. IFN-γ (2 ng/ml) was added after infection or pretreatment with the 19-kDa lipoprotein, and alveolar macrophages were cultured for an additional 24, 48, and 72 h in the presence of IFN-γ. The expression of MHC-II was measured by flow cytometry, and histograms representative of four independent experiments are shown. IFN-γ-induced MHC-II expression on BCG-infected or 19-kDa-lipoprotein-treated macrophages (black lines) is compared to control treated cells (shaded) after 48 and 72 h of IFN-γ exposure. Isotype control staining for both treatment groups is shown with overlapping thin lines. (E to F) Alveolar macrophages were isolated, cultured overnight, and then treated without (medium) or with the 19-kDa lipoprotein (500 ng/ml) for 24 h. After 24 h, fresh 19-kDa lipoprotein was added along with IFN-γ (2 ng/ml), and cells were incubated for an additional 48 h. CIITA and MHC-II mRNA expression was measured by real-time PCR and normalized to GAPDH expression. Data from a representative experiment ( n = 3) are shown. Decreases in CIITA and MHC-II mRNA expression were analyzed by using a paired Student's t test, and significant differences are designated with asterisks ( P ≤ 0.05).

    Article Snippet: M. bovis BCG Connaught (ATCC 35745) was grown at 37°C for 18 to 21 days in Proskeur-Beck medium (pH 7.0) containing l -asparagine (5.0 g/liter), potassium dihydrogen phosphate (5 g/liter), sesquimagnesium citrate (1.5 g/liter), dipotassium sulfate (0.5 g/liter), d -glucose (30 g/liter), and Tween-80 (0.05%).

    Techniques: Infection, Expressing, Cell Culture, Flow Cytometry, Cytometry, Staining, Isolation, Incubation, Real-time Polymerase Chain Reaction

    Phagocytosis of M. bovis BCG by alveolar macrophages. Alveolar macrophages were incubated overnight with fluos-BCG at a low MOI (3 to 5) and analyzed by flow cytometry. (A) A representative histogram shows phagocytosis of fluos-BCG. (B) MHC-II and B7.1 expression on fluos-BCG-positive cells (region R1, panel A) from a representative experiment reveals similar uptake of bacteria by both MHC-II + and MHC-II − B7.1 + alveolar macrophages.

    Journal: Infection and Immunity

    Article Title: Inhibition of Major Histocompatibility Complex II Expression and Antigen Processing in Murine Alveolar Macrophages by Mycobacterium bovis BCG and the 19-Kilodalton Mycobacterial Lipoprotein

    doi: 10.1128/IAI.72.4.2101-2110.2004

    Figure Lengend Snippet: Phagocytosis of M. bovis BCG by alveolar macrophages. Alveolar macrophages were incubated overnight with fluos-BCG at a low MOI (3 to 5) and analyzed by flow cytometry. (A) A representative histogram shows phagocytosis of fluos-BCG. (B) MHC-II and B7.1 expression on fluos-BCG-positive cells (region R1, panel A) from a representative experiment reveals similar uptake of bacteria by both MHC-II + and MHC-II − B7.1 + alveolar macrophages.

    Article Snippet: M. bovis BCG Connaught (ATCC 35745) was grown at 37°C for 18 to 21 days in Proskeur-Beck medium (pH 7.0) containing l -asparagine (5.0 g/liter), potassium dihydrogen phosphate (5 g/liter), sesquimagnesium citrate (1.5 g/liter), dipotassium sulfate (0.5 g/liter), d -glucose (30 g/liter), and Tween-80 (0.05%).

    Techniques: Incubation, Flow Cytometry, Cytometry, Expressing

    iNOS inhibition reverses the DCP-mediated antimycobacterial effects. (A) The addition of the iNOS inhibitor ( l -NMMA monoacetate) markedly reversed the DCP-mediated inhibition of intracellular M. bovis BCG growth. **, P = 0.0022 for untreated infected

    Journal: Infection and Immunity

    Article Title: Dipterinyl Calcium Pentahydrate Inhibits Intracellular Mycobacterial Growth in Human Monocytes via the C-C Chemokine MIP-1? and Nitric Oxide

    doi: 10.1128/IAI.01393-12

    Figure Lengend Snippet: iNOS inhibition reverses the DCP-mediated antimycobacterial effects. (A) The addition of the iNOS inhibitor ( l -NMMA monoacetate) markedly reversed the DCP-mediated inhibition of intracellular M. bovis BCG growth. **, P = 0.0022 for untreated infected

    Article Snippet: Mycobacterium bovis BCG Connaught strain (ATCC 35745; TMC 1030) was grown in suspension with constant gentle rotation in roller bottles containing Middlebrook 7H9 broth (BD Difco; 271310) supplemented with 10% Middlebrook albumin, dextrose, and catalase (ADC) enrichment (BD Biosciences; 211887) and 0.05% Tween 80 (Sigma-Aldrich).

    Techniques: Inhibition, Infection

    TLR2-mediated CCL5 production by epithelial cells involves calcineurin. HEK and TLR2 cells were pretreated with FK-506 (100 ng/ml) for 1 h. After incubation, cells were either left uninfected or stimulated with M. bovis BCG for a further 24 h. Culture

    Journal:

    Article Title: Mycobacterium bovis Bacillus Calmette-Gu?rin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-?B and -Jun N-Terminal Kinase Signaling Pathways ▿

    doi: 10.1128/CVI.00368-07

    Figure Lengend Snippet: TLR2-mediated CCL5 production by epithelial cells involves calcineurin. HEK and TLR2 cells were pretreated with FK-506 (100 ng/ml) for 1 h. After incubation, cells were either left uninfected or stimulated with M. bovis BCG for a further 24 h. Culture

    Article Snippet: M. bovis BCG (ATCC 35733) was obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Incubation

    M. bovis BCG activates NF-κB in epithelial cells. (A) Cells were infected with M. bovis BCG for the indicated times, and the levels of cytosolic and nuclear p65 and p50 were determined by immunoblotting with p65- and p50-specific antibodies, respectively.

    Journal:

    Article Title: Mycobacterium bovis Bacillus Calmette-Gu?rin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-?B and -Jun N-Terminal Kinase Signaling Pathways ▿

    doi: 10.1128/CVI.00368-07

    Figure Lengend Snippet: M. bovis BCG activates NF-κB in epithelial cells. (A) Cells were infected with M. bovis BCG for the indicated times, and the levels of cytosolic and nuclear p65 and p50 were determined by immunoblotting with p65- and p50-specific antibodies, respectively.

    Article Snippet: M. bovis BCG (ATCC 35733) was obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Infection

    Compared to negative controls, TLR2-expressing cells show increased JNK activation in response to infection by M. bovis BCG. HEK and TLR2 cells were infected with M. bovis BCG at an MOI of 3:1. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis

    Journal:

    Article Title: Mycobacterium bovis Bacillus Calmette-Gu?rin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-?B and -Jun N-Terminal Kinase Signaling Pathways ▿

    doi: 10.1128/CVI.00368-07

    Figure Lengend Snippet: Compared to negative controls, TLR2-expressing cells show increased JNK activation in response to infection by M. bovis BCG. HEK and TLR2 cells were infected with M. bovis BCG at an MOI of 3:1. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis

    Article Snippet: M. bovis BCG (ATCC 35733) was obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Expressing, Activation Assay, Infection, Polyacrylamide Gel Electrophoresis

    TLR2 expression enhances CCL2 and CCL5 production from M. bovis BCG-infected cells. HEK293 cells that do not express TLR2 (HEK) and HEK293 cells stably transfected with human TLR2 (TLR2) were either left uninfected or stimulated with M. bovis BCG at an

    Journal:

    Article Title: Mycobacterium bovis Bacillus Calmette-Gu?rin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-?B and -Jun N-Terminal Kinase Signaling Pathways ▿

    doi: 10.1128/CVI.00368-07

    Figure Lengend Snippet: TLR2 expression enhances CCL2 and CCL5 production from M. bovis BCG-infected cells. HEK293 cells that do not express TLR2 (HEK) and HEK293 cells stably transfected with human TLR2 (TLR2) were either left uninfected or stimulated with M. bovis BCG at an

    Article Snippet: M. bovis BCG (ATCC 35733) was obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Expressing, Infection, Stable Transfection, Transfection

    M. bovis BCG-mediated CCL2 and CCL5 secretion is impaired by JNK inhibitors. HEK and TLR2 cells were pretreated with SP600125 (10 μM) or curcumin (5 μM) for 1 h and then infected with M. bovis BCG for a further 24 h. Culture supernatants

    Journal:

    Article Title: Mycobacterium bovis Bacillus Calmette-Gu?rin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-?B and -Jun N-Terminal Kinase Signaling Pathways ▿

    doi: 10.1128/CVI.00368-07

    Figure Lengend Snippet: M. bovis BCG-mediated CCL2 and CCL5 secretion is impaired by JNK inhibitors. HEK and TLR2 cells were pretreated with SP600125 (10 μM) or curcumin (5 μM) for 1 h and then infected with M. bovis BCG for a further 24 h. Culture supernatants

    Article Snippet: M. bovis BCG (ATCC 35733) was obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Infection

    Bacterial growth in lungs (A and B) and spleens (C and D) after low-dose aerosol infection of 6–8-week-old C57BL/6 mice with wild-type H37Rv ([unk]), H37Rv:ΔRD1 (■), or BCG-Russia (▲). A and C, Early-phase infection and dissemination. B and D, Progression of infection through 21 weeks. Data at each time point are the mean and SD of 15 mice per strain from 3 separate infections.

    Journal: The Journal of infectious diseases

    Article Title: Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Gu?rin Attenuation

    doi:

    Figure Lengend Snippet: Bacterial growth in lungs (A and B) and spleens (C and D) after low-dose aerosol infection of 6–8-week-old C57BL/6 mice with wild-type H37Rv ([unk]), H37Rv:ΔRD1 (■), or BCG-Russia (▲). A and C, Early-phase infection and dissemination. B and D, Progression of infection through 21 weeks. Data at each time point are the mean and SD of 15 mice per strain from 3 separate infections.

    Article Snippet: H37Rv:ΔRD1 clones were compared with virulent parent strain H37Rv and with attenuated BCG-Russia (ATCC 35740), which was chosen because it is potentially more similar to the original BCG than other extant BCG strains [ ].

    Techniques: Infection, Mouse Assay

    Bacterial growth and cytotoxicity in a human macrophage THP-1 cell line (A and B) and growth in human peripheral blood mononuclear cell (PBMC)–derived macrophages (C) after infection by H37Rv ([unk]), H37Rv:ΔRD1 (■), or BCG-Russia (▲). Data for each time point are the mean and SD of 4 infections in panels A and B and of a representative infection (of 4) in panel C. Bacteria were quantitated by luciferase assay (A) or by plating for colony-forming units (C). Cytotoxicity is indicated by the decline in macrophage metabolism over the course of infection (B).

    Journal: The Journal of infectious diseases

    Article Title: Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Gu?rin Attenuation

    doi:

    Figure Lengend Snippet: Bacterial growth and cytotoxicity in a human macrophage THP-1 cell line (A and B) and growth in human peripheral blood mononuclear cell (PBMC)–derived macrophages (C) after infection by H37Rv ([unk]), H37Rv:ΔRD1 (■), or BCG-Russia (▲). Data for each time point are the mean and SD of 4 infections in panels A and B and of a representative infection (of 4) in panel C. Bacteria were quantitated by luciferase assay (A) or by plating for colony-forming units (C). Cytotoxicity is indicated by the decline in macrophage metabolism over the course of infection (B).

    Article Snippet: H37Rv:ΔRD1 clones were compared with virulent parent strain H37Rv and with attenuated BCG-Russia (ATCC 35740), which was chosen because it is potentially more similar to the original BCG than other extant BCG strains [ ].

    Techniques: Derivative Assay, Infection, Luciferase

    Lung histopathology after low-dose aerosol infection by H37Rv (A, D, and G), H37Rv:ΔRD1 (B, E, and H), or BCG-Russia (C, F, and I). At each time point, the left lung was removed, inflated, fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylineosin. Photographs shown are representative of at least 15 mice/strain. A, B, and C, Differences in overall lung involvement (original magnification, ×10) at week 6 after infection. D, E, and F, Differences in granuloma formation (original magnification, ×50; at week 6 after infection). D, Well-organized granuloma with tight rim of lymphocytes surrounding central core of histiocytes in a mouse infected with wild-type MTB. E and F, Areas of greatest inflammation in the H37:ΔRD1-infected (E) or BCG-Russia–infected (F) lung, consisting only of mild perivascular lymphocyte cuffs. G, H, and I, Differences in lung inflammation at week 21 after infection (original magnification, ×10).

    Journal: The Journal of infectious diseases

    Article Title: Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Gu?rin Attenuation

    doi:

    Figure Lengend Snippet: Lung histopathology after low-dose aerosol infection by H37Rv (A, D, and G), H37Rv:ΔRD1 (B, E, and H), or BCG-Russia (C, F, and I). At each time point, the left lung was removed, inflated, fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylineosin. Photographs shown are representative of at least 15 mice/strain. A, B, and C, Differences in overall lung involvement (original magnification, ×10) at week 6 after infection. D, E, and F, Differences in granuloma formation (original magnification, ×50; at week 6 after infection). D, Well-organized granuloma with tight rim of lymphocytes surrounding central core of histiocytes in a mouse infected with wild-type MTB. E and F, Areas of greatest inflammation in the H37:ΔRD1-infected (E) or BCG-Russia–infected (F) lung, consisting only of mild perivascular lymphocyte cuffs. G, H, and I, Differences in lung inflammation at week 21 after infection (original magnification, ×10).

    Article Snippet: H37Rv:ΔRD1 clones were compared with virulent parent strain H37Rv and with attenuated BCG-Russia (ATCC 35740), which was chosen because it is potentially more similar to the original BCG than other extant BCG strains [ ].

    Techniques: Histopathology, Infection, Staining, Mouse Assay

    Anhydrotetracycline-inducible expression of Myc-tagged M. tuberculosis σ factors. (A) Structure of the plasmids used for expression of σ factors. OriE, replication origin in E. coli ; OriM, pAL5000 mycobacterial replication determinant; Hyg, hygromycin resistance gene; TetR, Tn 10 Tet repressor; pUV15-tetOrm, strong TetR-controlled mycobacterial promoter. (B) Time-dependent induction of Myc-tagged σ A in M. bovis BCG-Russia. Samples were collected at 2, 4, 6, 8, 12, and 24 h after addition of 50 ng/ml anhydrotetracycline to the growth medium. Protein extracts were prepared and used for immunoblotting with the Myc 9E10 antibody. Lane C contained recombinant Myc-tagged σ A .

    Journal: Journal of Bacteriology

    Article Title: Identification of Mycobacterial ? Factor Binding Sites by Chromatin Immunoprecipitation Assays ▿

    doi: 10.1128/JB.01371-06

    Figure Lengend Snippet: Anhydrotetracycline-inducible expression of Myc-tagged M. tuberculosis σ factors. (A) Structure of the plasmids used for expression of σ factors. OriE, replication origin in E. coli ; OriM, pAL5000 mycobacterial replication determinant; Hyg, hygromycin resistance gene; TetR, Tn 10 Tet repressor; pUV15-tetOrm, strong TetR-controlled mycobacterial promoter. (B) Time-dependent induction of Myc-tagged σ A in M. bovis BCG-Russia. Samples were collected at 2, 4, 6, 8, 12, and 24 h after addition of 50 ng/ml anhydrotetracycline to the growth medium. Protein extracts were prepared and used for immunoblotting with the Myc 9E10 antibody. Lane C contained recombinant Myc-tagged σ A .

    Article Snippet: M. bovis BCG-Russia (ATCC 35740) was grown at 37°C in Middlebrook 7H9 medium supplemented with albumin-dextrose-saline, 0.05% Tween 80, and 10 μg/ml cycloheximide.

    Techniques: Expressing, Recombinant

    Metabolic network of the central metabolism of Mycobacterium bovis BCG. Glycolysis/gluconeogenesis (green), oxidative pentose phosphate pathway (orange), tricarboxylic acid cycle (TCA, pink), anaplerotic reactions (blue), and biosynthesis (grey). Standard abbreviations are used for the amino acids. Metabolite abbreviations: ACCOA, acetyl-CoA; ACE, acetate; CHO, chorismate; E4P, d -erythrose 4-phosphate; F6P, d -fructose 6-phosphate; FBP, d -fructose 1,6-bisphosphate; FUM, fumarate; G6P, d -glucose 6-phosphate; GA3P, glyceraldehyde 3-phosphate; GLX, glyoxylate; GLYC, glycerol; ICIT, isocitrate/citrate; KIV, 2-oxoisovalerate; MALOAA, l -malate-oxaloacetate; OXG, 2-oxoglutarate; R5P,α- d -ribose 5-phosphate/ l -ribulose 5-phosphate/ l -xylulose 5-phosphate; PEP, phosphoenolpyruvate; PGA, 2-phospho- d -glycerate/3-phospho- d -glycerate; PYR, pyruvate; S7P, sedoheptulose 7-phosphate; SUC, succinate; SUCCOA, succinyl-CoA. Enzyme abbreviations: CS: citrate synthase; ENO: enolase; FBA: fructose-bisphosphate adolase; FBP: fructose-bisphosphatase; FUM:fumurase; GAPA: glyceraldehyde 3-phosphate dehydrogenase; GND: 6-phosphogluconate dehydrogenase; ICL: isocitrate lyase; ICDH: isocitrate dehydrogenase NADP-dependent; KOR/KGD: α-ketoglutarate ferredoxin oxidoreductase/α-ketoglutarate decarboxylase;MEZ/PCA: malic enzyme/pyruvate carboxylase: MDH: malate dehydrogenase; MS: malate synthase; PCK: phosphoenolpyruvate carboxykinase; PDH: pyruvate dehydrogenase; PGI: glucose phosphate isomerase; PYK: pyruvate kinase;SDH: succinate dehydrogenase; SCS: Succinyl CoA synthetase; TAL: transaldolase; TKT1/2: transketolase. Explicit names are also given in Table S6 . The picture was generated with Omix, an editor for biochemical networks and visualization tool [ http://www.13cflux.net/omix ].

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: Metabolic network of the central metabolism of Mycobacterium bovis BCG. Glycolysis/gluconeogenesis (green), oxidative pentose phosphate pathway (orange), tricarboxylic acid cycle (TCA, pink), anaplerotic reactions (blue), and biosynthesis (grey). Standard abbreviations are used for the amino acids. Metabolite abbreviations: ACCOA, acetyl-CoA; ACE, acetate; CHO, chorismate; E4P, d -erythrose 4-phosphate; F6P, d -fructose 6-phosphate; FBP, d -fructose 1,6-bisphosphate; FUM, fumarate; G6P, d -glucose 6-phosphate; GA3P, glyceraldehyde 3-phosphate; GLX, glyoxylate; GLYC, glycerol; ICIT, isocitrate/citrate; KIV, 2-oxoisovalerate; MALOAA, l -malate-oxaloacetate; OXG, 2-oxoglutarate; R5P,α- d -ribose 5-phosphate/ l -ribulose 5-phosphate/ l -xylulose 5-phosphate; PEP, phosphoenolpyruvate; PGA, 2-phospho- d -glycerate/3-phospho- d -glycerate; PYR, pyruvate; S7P, sedoheptulose 7-phosphate; SUC, succinate; SUCCOA, succinyl-CoA. Enzyme abbreviations: CS: citrate synthase; ENO: enolase; FBA: fructose-bisphosphate adolase; FBP: fructose-bisphosphatase; FUM:fumurase; GAPA: glyceraldehyde 3-phosphate dehydrogenase; GND: 6-phosphogluconate dehydrogenase; ICL: isocitrate lyase; ICDH: isocitrate dehydrogenase NADP-dependent; KOR/KGD: α-ketoglutarate ferredoxin oxidoreductase/α-ketoglutarate decarboxylase;MEZ/PCA: malic enzyme/pyruvate carboxylase: MDH: malate dehydrogenase; MS: malate synthase; PCK: phosphoenolpyruvate carboxykinase; PDH: pyruvate dehydrogenase; PGI: glucose phosphate isomerase; PYK: pyruvate kinase;SDH: succinate dehydrogenase; SCS: Succinyl CoA synthetase; TAL: transaldolase; TKT1/2: transketolase. Explicit names are also given in Table S6 . The picture was generated with Omix, an editor for biochemical networks and visualization tool [ http://www.13cflux.net/omix ].

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques: Mass Spectrometry, Generated

    Continuous culture of M. bovis BCG and ▵ icl 1 at a dilution rate of 0.03 h −1 (t d = 23.1 h). A 10% late log phase culture of M. bovis BCG (black line) or ▵ icl 1 (red line) was inoculated into the bioreactor. The cultures were operated as batch until day 4. Continuous culture was then started at a dilution rate of 0.03 h −1 . Samples were removed and assayed for biomass (circles). CO 2 production in the waste gas was measured using a dual tandem sensor gas analyser (squares). The results are representative of two experiments.

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: Continuous culture of M. bovis BCG and ▵ icl 1 at a dilution rate of 0.03 h −1 (t d = 23.1 h). A 10% late log phase culture of M. bovis BCG (black line) or ▵ icl 1 (red line) was inoculated into the bioreactor. The cultures were operated as batch until day 4. Continuous culture was then started at a dilution rate of 0.03 h −1 . Samples were removed and assayed for biomass (circles). CO 2 production in the waste gas was measured using a dual tandem sensor gas analyser (squares). The results are representative of two experiments.

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques:

    Continuous culture of M. bovis BCG and ▵ icl 1 at a dilution rate of 0.01 h −1 (t d = 69.3 h). A 10% late log phase culture of M. bovis BCG (A) and ▵ icl 1 (B) was inoculated into the bioreactor. The cultures were operated as batch until day 4. Continuous culture was then started at a dilution rate of 0.01 h −1 Samples were removed and assayed for biomass (red line). CO 2 production in the waste gas was measured using a dual tandem sensor gas analyser (green line) and filtered supernatants were assayed for glycerol (blue line). The results are representative of two experiments.

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: Continuous culture of M. bovis BCG and ▵ icl 1 at a dilution rate of 0.01 h −1 (t d = 69.3 h). A 10% late log phase culture of M. bovis BCG (A) and ▵ icl 1 (B) was inoculated into the bioreactor. The cultures were operated as batch until day 4. Continuous culture was then started at a dilution rate of 0.01 h −1 Samples were removed and assayed for biomass (red line). CO 2 production in the waste gas was measured using a dual tandem sensor gas analyser (green line) and filtered supernatants were assayed for glycerol (blue line). The results are representative of two experiments.

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques:

    Metabolic flux map corresponding to solution A for M. bovis BCG in glycerol limited continuous culture at slow growth rate (t d = 69 h). Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Mass spectral data of proteinogenic amino acids from steady state chemostat cultures grown on a mixture of 20% [U-13C] glycerol and 80% unlabeled glycerol and the physiological data of Table 1 were used to generate the two alternative flux estimations. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes ( Table S5 ) are indicated on each flux arrow. The abbreviations are as in Figure 4 .

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: Metabolic flux map corresponding to solution A for M. bovis BCG in glycerol limited continuous culture at slow growth rate (t d = 69 h). Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Mass spectral data of proteinogenic amino acids from steady state chemostat cultures grown on a mixture of 20% [U-13C] glycerol and 80% unlabeled glycerol and the physiological data of Table 1 were used to generate the two alternative flux estimations. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes ( Table S5 ) are indicated on each flux arrow. The abbreviations are as in Figure 4 .

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques:

    Batch cultivation of M. bovis BCG and ▵ icl 1 in a bioreactor. A 10% late log phase culture of M. bovis BCG (black line) or ▵ icl 1 (red line) was inoculated into the bioreactor and optical density at 600 nm was monitored for 4 d prior to switching to chemostat mode. Average values and standard deviations are shown from two independent experiments.

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: Batch cultivation of M. bovis BCG and ▵ icl 1 in a bioreactor. A 10% late log phase culture of M. bovis BCG (black line) or ▵ icl 1 (red line) was inoculated into the bioreactor and optical density at 600 nm was monitored for 4 d prior to switching to chemostat mode. Average values and standard deviations are shown from two independent experiments.

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques:

    In vivo flux distribution of M. bovis BCG in glycerol limited continuous culture at fast growth rate (t d = 23 h). Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes ( Table S5 ) are indicated on each flux arrow. The abbreviations are as in Figure 4 .

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: In vivo flux distribution of M. bovis BCG in glycerol limited continuous culture at fast growth rate (t d = 23 h). Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes ( Table S5 ) are indicated on each flux arrow. The abbreviations are as in Figure 4 .

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques: In Vivo

    Metabolic flux map B for M. bovis BCG in glycerol limited continuous culture at slow growth rate (t d = 69 h). Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Mass spectral data of proteinogenic amino acids from steady state chemostat cultures grown on a mixture of 20% [U-13C] glycerol and 80% unlabeled glycerol and the physiological data of Table 1 were used to generate the two alternative flux estimations. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes ( Table S5 ) are indicated on each flux arrow. The abbreviations are as in Figure 4 .

    Journal: PLoS Pathogens

    Article Title: 13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

    doi: 10.1371/journal.ppat.1002091

    Figure Lengend Snippet: Metabolic flux map B for M. bovis BCG in glycerol limited continuous culture at slow growth rate (t d = 69 h). Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Mass spectral data of proteinogenic amino acids from steady state chemostat cultures grown on a mixture of 20% [U-13C] glycerol and 80% unlabeled glycerol and the physiological data of Table 1 were used to generate the two alternative flux estimations. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes ( Table S5 ) are indicated on each flux arrow. The abbreviations are as in Figure 4 .

    Article Snippet: Bacterial strains and growth conditions M. bovis BCG strain (ATCC 35748) or Mycobacterium tuberculosis (H37Rv) was cultured in a 2- l bioreactor (Adaptive Biosystem Voyager) under aerobic conditions and at pH 6.6 as previously described .

    Techniques:

    Functional characterization of significantly altered proteins in THP-1 cells infected with MTBC strain H37Rv , M. bovis , or BCG. (A) Top canonical pathways, (B) diseases and disorders, (C) molecular and cellular functions, (D) physiological system development and functions, and (E) toxicity functions.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Comparative Proteomics Analysis of Human Macrophages Infected with Virulent Mycobacterium bovis

    doi: 10.3389/fcimb.2017.00065

    Figure Lengend Snippet: Functional characterization of significantly altered proteins in THP-1 cells infected with MTBC strain H37Rv , M. bovis , or BCG. (A) Top canonical pathways, (B) diseases and disorders, (C) molecular and cellular functions, (D) physiological system development and functions, and (E) toxicity functions.

    Article Snippet: Bacterial strains and culture conditions The M. tb reference strain H37Rv (ATCC 27294), BCG Tokyo strain (ATCC 35737), and M. bovis (ATCC 19210) were kindly provided by Dr. Chuanyou Li, Beijing Tuberculosis & Thoracic Tumor Research Institute (Beijing, China).

    Techniques: Functional Assay, Infection

    Real-time RT-PCR analysis of significantly altered proteins in THP-1 cells infected by MTBC (10 MOI) or a mock. (A) BCG in black bars, (B) H37Rv in gray bars, (C) M. bovis in dark gray bars.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Comparative Proteomics Analysis of Human Macrophages Infected with Virulent Mycobacterium bovis

    doi: 10.3389/fcimb.2017.00065

    Figure Lengend Snippet: Real-time RT-PCR analysis of significantly altered proteins in THP-1 cells infected by MTBC (10 MOI) or a mock. (A) BCG in black bars, (B) H37Rv in gray bars, (C) M. bovis in dark gray bars.

    Article Snippet: Bacterial strains and culture conditions The M. tb reference strain H37Rv (ATCC 27294), BCG Tokyo strain (ATCC 35737), and M. bovis (ATCC 19210) were kindly provided by Dr. Chuanyou Li, Beijing Tuberculosis & Thoracic Tumor Research Institute (Beijing, China).

    Techniques: Quantitative RT-PCR, Infection

    The detailed view of the top-score networks . Red indicates significantly up-regulated proteins, and green indicates significantly down-regulated proteins. White indicates the proteins that were not identified in our data. The color depth reflects the fold-change of proteins. The shapes are indicative of the molecular class. Lines with arrows connecting between the molecules indicate molecular relationships. Solid lines indicate direct interactions, and dashed lines indicate indirect interactions. (A) The top-score IPA network of BCG infection: cellular development, cellular growth and proliferation, cell death and survival, (B) the top two IPA networks of H37Rv infection: cellular movement, hematological system development and function, and immune cell trafficking (left); cancer, cell death, and survival, and cellular movement (right), (C) the top two IPA networks of M. bovis infection: free radical scavenging, hereditary disorder, and immunological disease (left); cell death and survival, organismal injury and abnormalities, and tissue morphology (right). Additional networks are depicted in Table S4 .

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Comparative Proteomics Analysis of Human Macrophages Infected with Virulent Mycobacterium bovis

    doi: 10.3389/fcimb.2017.00065

    Figure Lengend Snippet: The detailed view of the top-score networks . Red indicates significantly up-regulated proteins, and green indicates significantly down-regulated proteins. White indicates the proteins that were not identified in our data. The color depth reflects the fold-change of proteins. The shapes are indicative of the molecular class. Lines with arrows connecting between the molecules indicate molecular relationships. Solid lines indicate direct interactions, and dashed lines indicate indirect interactions. (A) The top-score IPA network of BCG infection: cellular development, cellular growth and proliferation, cell death and survival, (B) the top two IPA networks of H37Rv infection: cellular movement, hematological system development and function, and immune cell trafficking (left); cancer, cell death, and survival, and cellular movement (right), (C) the top two IPA networks of M. bovis infection: free radical scavenging, hereditary disorder, and immunological disease (left); cell death and survival, organismal injury and abnormalities, and tissue morphology (right). Additional networks are depicted in Table S4 .

    Article Snippet: Bacterial strains and culture conditions The M. tb reference strain H37Rv (ATCC 27294), BCG Tokyo strain (ATCC 35737), and M. bovis (ATCC 19210) were kindly provided by Dr. Chuanyou Li, Beijing Tuberculosis & Thoracic Tumor Research Institute (Beijing, China).

    Techniques: Indirect Immunoperoxidase Assay, Infection

    Skin test reactivity of multiantigen cocktails in guinea pigs immunized with M. bovis BCG (solid symbols) and with M. avium (open symbols). Six guinea pigs were sensitized and skin tested as described in Materials and Methods. In this set of experiments, animals were skin tested with 10 TU of PPD and increasing amounts (0.5 to 8 μg) of multiantigen cocktails. Results are expressed as means of the diameters of erythema measured 24 h after antigen injection. Cocktail A, four M. tuberculosis complex-specific antigens (MPT63, MPT64, MTC28, and MPT70); cocktail B, four cross-reactive antigens (MPT51, Ag85B, MPT32, and KatG); cocktail C, eight-antigen cocktail (antigens in cocktails A plus B). ■, □, M. bovis PPD; ▴, ▵, M. avium PPD; •, ○, cocktails of purified antigens.

    Journal: Infection and Immunity

    Article Title: Use of Mycobacterium tuberculosis Complex-Specific Antigen Cocktails for a Skin Test Specific for Tuberculosis

    doi:

    Figure Lengend Snippet: Skin test reactivity of multiantigen cocktails in guinea pigs immunized with M. bovis BCG (solid symbols) and with M. avium (open symbols). Six guinea pigs were sensitized and skin tested as described in Materials and Methods. In this set of experiments, animals were skin tested with 10 TU of PPD and increasing amounts (0.5 to 8 μg) of multiantigen cocktails. Results are expressed as means of the diameters of erythema measured 24 h after antigen injection. Cocktail A, four M. tuberculosis complex-specific antigens (MPT63, MPT64, MTC28, and MPT70); cocktail B, four cross-reactive antigens (MPT51, Ag85B, MPT32, and KatG); cocktail C, eight-antigen cocktail (antigens in cocktails A plus B). ■, □, M. bovis PPD; ▴, ▵, M. avium PPD; •, ○, cocktails of purified antigens.

    Article Snippet: M. bovis BCG Japanese ATCC 35737 and M. avium ATCC 25291 were obtained from the American Type Culture Collection.

    Techniques: Injection, Purification

    Skin test reactivity of purified recombinant antigens of M. tuberculosis tested singly and in combinations in guinea pigs immunized with M. bovis BCG. Six guinea pigs were sensitized as described in Materials and Methods. Five weeks after sensitization, animals were intradermally injected with 10 TU of PPD and 2 μg of purified recombinant antigens, singly or in combination. Results are expressed as the means (plus standard deviations) of the diameters of erythema measured 24 h after antigen injection. Ags 1–6, six antigens injected singly (1, MPT63; 2, MPT64; 3, MTC28; 4, MPT32; 5, MPT51; 6, 38 kDa); Combi1-2, two-antigen cocktail (antigens 1 and 2); Combi1-4, four-antigen cocktail (antigens 1 through 4); Combi1-6, six-antigen cocktail (antigens 1 through 6).

    Journal: Infection and Immunity

    Article Title: Use of Mycobacterium tuberculosis Complex-Specific Antigen Cocktails for a Skin Test Specific for Tuberculosis

    doi:

    Figure Lengend Snippet: Skin test reactivity of purified recombinant antigens of M. tuberculosis tested singly and in combinations in guinea pigs immunized with M. bovis BCG. Six guinea pigs were sensitized as described in Materials and Methods. Five weeks after sensitization, animals were intradermally injected with 10 TU of PPD and 2 μg of purified recombinant antigens, singly or in combination. Results are expressed as the means (plus standard deviations) of the diameters of erythema measured 24 h after antigen injection. Ags 1–6, six antigens injected singly (1, MPT63; 2, MPT64; 3, MTC28; 4, MPT32; 5, MPT51; 6, 38 kDa); Combi1-2, two-antigen cocktail (antigens 1 and 2); Combi1-4, four-antigen cocktail (antigens 1 through 4); Combi1-6, six-antigen cocktail (antigens 1 through 6).

    Article Snippet: M. bovis BCG Japanese ATCC 35737 and M. avium ATCC 25291 were obtained from the American Type Culture Collection.

    Techniques: Purification, Recombinant, Injection

    Survival curves of mice infected with M. tuberculosis or M. bovis BCG Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.

    Journal: Infection and Immunity

    Article Title: Role of Interleukin-18 (IL-18) in Mycobacterial Infection in IL-18-Gene-Disrupted Mice

    doi:

    Figure Lengend Snippet: Survival curves of mice infected with M. tuberculosis or M. bovis BCG Pasteur. IL-18-KO and WT mice were infected i.v. with 10 6 CFU of the Kurono or BCG Pasteur strain. Data presented are from two separate experiments with 10 mice in each group.

    Article Snippet: The virulent Kurono strain (ATCC 358121) and the avirulent strain Mycobacterium bovis BCG Pasteur (ATCC 27289) of M. tuberculosis were grown in Middlebrook 7H9 medium (Difco) to the mid-log phase ( ).

    Techniques: Mouse Assay, Infection

    qPCR amplification curves and standard curves of the serial dilution using (a) M. tuberculosis H37Rv; (b) M. bovis BCG Pasteur ATCC 35734; (c) M. microti ATCC 19422; and (d) M. caprae ZH 22914 for high‐resolution melting (HRM) assay 2

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: qPCR amplification curves and standard curves of the serial dilution using (a) M. tuberculosis H37Rv; (b) M. bovis BCG Pasteur ATCC 35734; (c) M. microti ATCC 19422; and (d) M. caprae ZH 22914 for high‐resolution melting (HRM) assay 2

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Serial Dilution, HRM Assay

    Differentiation of MTBC based on eight different single nucleotide polymorphisms (SNPs). Numbers represent the position of the SNP in relation to the start codon of gyrB and gyrA , respectively. (a) Six SNPs on gyrB are represented. HRM assay 1 (Landolt et al., 2019 ) allows the distinction between M. tuberculosis / M. canettii / M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti / M. canettii (rare subtype) and M. bovis / M. bovis BCG/ M. caprae /rare M. caprae / M. bovis ecotypes. HRM assay 2 can differentiate between M. tuberculosis / M. canettii , M. canettii (rare subtype), M. africanum / M. orygis / M. pinnipedii /Clade A1/ M. microti / M. caprae /rare M. caprae / M. bovis ecotypes, and M. bovis / M. bovis BCG. Combining the results of HRM assays 1 and 2, six groups can be distinguished: M. canettii (rare subtype), M. tuberculosis / M. canettii , M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti , M. caprae /rare M. caprae / M. bovis ecotypes, and M. bovis / M. bovis BCG. (b) Two SNPs on positions 1,323 and 1,359 of gyrA are illustrated indicating the differentiation between M. bovis , M. bovis BCG, and rare M. caprae / M. bovis ecotype I. *Rare subtype, highly recombinogenic strain, intrinsic pyrazinamide (PZA) resistance. **Intrinsic PZA resistance. ***No intrinsic PZA resistance. ****Intrinsic PZA and cycloserine resistance

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: Differentiation of MTBC based on eight different single nucleotide polymorphisms (SNPs). Numbers represent the position of the SNP in relation to the start codon of gyrB and gyrA , respectively. (a) Six SNPs on gyrB are represented. HRM assay 1 (Landolt et al., 2019 ) allows the distinction between M. tuberculosis / M. canettii / M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti / M. canettii (rare subtype) and M. bovis / M. bovis BCG/ M. caprae /rare M. caprae / M. bovis ecotypes. HRM assay 2 can differentiate between M. tuberculosis / M. canettii , M. canettii (rare subtype), M. africanum / M. orygis / M. pinnipedii /Clade A1/ M. microti / M. caprae /rare M. caprae / M. bovis ecotypes, and M. bovis / M. bovis BCG. Combining the results of HRM assays 1 and 2, six groups can be distinguished: M. canettii (rare subtype), M. tuberculosis / M. canettii , M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti , M. caprae /rare M. caprae / M. bovis ecotypes, and M. bovis / M. bovis BCG. (b) Two SNPs on positions 1,323 and 1,359 of gyrA are illustrated indicating the differentiation between M. bovis , M. bovis BCG, and rare M. caprae / M. bovis ecotype I. *Rare subtype, highly recombinogenic strain, intrinsic pyrazinamide (PZA) resistance. **Intrinsic PZA resistance. ***No intrinsic PZA resistance. ****Intrinsic PZA and cycloserine resistance

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: HRM Assay

    qPCR amplification curves and standard curves of the serial dilution using (a) M. bovis BCG Pasteur ATCC 35734 and (b) M. bovis for high‐resolution melting (HRM) assay 3

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: qPCR amplification curves and standard curves of the serial dilution using (a) M. bovis BCG Pasteur ATCC 35734 and (b) M. bovis for high‐resolution melting (HRM) assay 3

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Serial Dilution, HRM Assay

    Representative high‐resolution melting graphs corresponding to one high‐resolution melting analysis of a subset of cultured samples ( n = 9) and clinical specimens ( n = 7) of HRM assay 3. Curves of tested samples previously identified as M. bovis BCG are shown in pink, cultured samples of M. bovis in red, and clinical specimen of M. bovis in orange. (a) Melting curves; (b) normalized plot; and (c) difference plot in relation to M. bovis BCG Pasteur ATCC 35734

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: Representative high‐resolution melting graphs corresponding to one high‐resolution melting analysis of a subset of cultured samples ( n = 9) and clinical specimens ( n = 7) of HRM assay 3. Curves of tested samples previously identified as M. bovis BCG are shown in pink, cultured samples of M. bovis in red, and clinical specimen of M. bovis in orange. (a) Melting curves; (b) normalized plot; and (c) difference plot in relation to M. bovis BCG Pasteur ATCC 35734

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: Cell Culture, HRM Assay

    Representative high‐resolution melting graphs corresponding to one high‐resolution melting analysis of a subset of cultured samples ( n = 22) of HRM assay 2. Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis / M. bovis BCG in red, and M. caprae in green. (a) Melting curves; (b) normalized plot; and (c) difference plot in relation to M. bovis BCG Pasteur ATCC 35734

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: Representative high‐resolution melting graphs corresponding to one high‐resolution melting analysis of a subset of cultured samples ( n = 22) of HRM assay 2. Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis / M. bovis BCG in red, and M. caprae in green. (a) Melting curves; (b) normalized plot; and (c) difference plot in relation to M. bovis BCG Pasteur ATCC 35734

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: Cell Culture, HRM Assay

    HRM assay 1 (Landolt et al., 2019 ) allows the distinction between M. tuberculosis / M. canettii / M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti / M. canettii (rare subtype), and M. bovis / M. bovis BCG/ M. caprae /rare M. caprae / M. bovis ecotypes. A combination of HRM assays 1 and 2 is leading to six groups ( M. tuberculosis / M. canettii , M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti , M. canettii (rare subtype), M. caprae /rare M. caprae / M. bovis ecotypes I and II, and M. bovis / M. bovis BCG). By performing HRM assay 3, M. bovis , M. bovis BCG, and rare M. caprae / M. bovis ecotype I can further be distinguished. 1. M. africanum not distinguishable in gyrA and gyrB from M. orygis , M. pinnipedii , Clade A1 (Dassie bacillus, M. mungi , Chimpanzee bacillus, M. suricattae ) (Brites et al., 2018 ). 2. Frequent subtype, intrinsic pyrazinamide (PZA) resistance. 3. Rare ecotypes, no intrinsic PZA resistance. 4. Rare subtype, highly recombinogenic

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: HRM assay 1 (Landolt et al., 2019 ) allows the distinction between M. tuberculosis / M. canettii / M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti / M. canettii (rare subtype), and M. bovis / M. bovis BCG/ M. caprae /rare M. caprae / M. bovis ecotypes. A combination of HRM assays 1 and 2 is leading to six groups ( M. tuberculosis / M. canettii , M. africanum / M. orygis / M. pinnipedii /Clade A1, M. microti , M. canettii (rare subtype), M. caprae /rare M. caprae / M. bovis ecotypes I and II, and M. bovis / M. bovis BCG). By performing HRM assay 3, M. bovis , M. bovis BCG, and rare M. caprae / M. bovis ecotype I can further be distinguished. 1. M. africanum not distinguishable in gyrA and gyrB from M. orygis , M. pinnipedii , Clade A1 (Dassie bacillus, M. mungi , Chimpanzee bacillus, M. suricattae ) (Brites et al., 2018 ). 2. Frequent subtype, intrinsic pyrazinamide (PZA) resistance. 3. Rare ecotypes, no intrinsic PZA resistance. 4. Rare subtype, highly recombinogenic

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: HRM Assay

    Representative high‐resolution melting graphs corresponding to one high‐resolution melting analysis of a subset of clinical specimens ( n = 18) for HRM assay 2. Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis / M. bovis BCG in red, and M. caprae in green. (a) Melting curves; (b) normalized plot; and (c) difference plot in relation to M. bovis BCG Pasteur ATCC 35734

    Journal: MicrobiologyOpen

    Article Title: Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members. Three‐reaction high‐resolution melting assay for rapid differentiation of Mycobacterium tuberculosis complex members

    doi: 10.1002/mbo3.919

    Figure Lengend Snippet: Representative high‐resolution melting graphs corresponding to one high‐resolution melting analysis of a subset of clinical specimens ( n = 18) for HRM assay 2. Curves of tested samples previously identified as M. tuberculosis are shown in yellow, M. microti in blue, M. bovis / M. bovis BCG in red, and M. caprae in green. (a) Melting curves; (b) normalized plot; and (c) difference plot in relation to M. bovis BCG Pasteur ATCC 35734

    Article Snippet: Additionally, for HRM assay 3, M. bovis BCG Tice ATCC 27289 was used.

    Techniques: HRM Assay

    Combined therapy of BCG and L-NAME exerts an antitumoral effect through modulation of MACs and fibroblasts in the tumor microenvironment. BCG directly induces fibroblast proliferation via MAPK and PI3K signaling pathways as well as alpha-SMA and collagen expression. BCG induces in MACs the production of NO and soluble factors including FGF-2 which induce fibroblast proliferation. BCG therapy increases collagen deposition and expression of alpha-SMA and FGF-2 in bladder tumors The treatment with L-NAME, improves the stimulation of fibroblasts by BCG.

    Journal: PLoS ONE

    Article Title: Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    doi: 10.1371/journal.pone.0013571

    Figure Lengend Snippet: Combined therapy of BCG and L-NAME exerts an antitumoral effect through modulation of MACs and fibroblasts in the tumor microenvironment. BCG directly induces fibroblast proliferation via MAPK and PI3K signaling pathways as well as alpha-SMA and collagen expression. BCG induces in MACs the production of NO and soluble factors including FGF-2 which induce fibroblast proliferation. BCG therapy increases collagen deposition and expression of alpha-SMA and FGF-2 in bladder tumors The treatment with L-NAME, improves the stimulation of fibroblasts by BCG.

    Article Snippet: To evaluate whether the MAPK or PI3K pathways were involved in BCG induction, NIH-3T3 cells were treated with BCG plus 10 and 20 µM of LY 294002 (Millipore) for 24 h or 10 and 50 uM of PD 98059 (Millipore) for 48 h. To evaluate NOS inhibitor activity on fibroblast viability, similar experiments were carried out in the presence of different concentrations of L-NAME (N-nitro-L-arginine-methyl-ester, Sigma).

    Techniques: Magnetic Cell Separation, Expressing

    L-NAME improves BCG-induced proliferation. NIH-3T3 fibroblast were treated with BCG (3×10 6 CFU/ml), L-NAME (2 mM) or BCG and L-NAME in presence of LY 294002 and PD 98059 for 24 and 48 h respectively. Cell viability was evaluated by MTS. a: p

    Journal: PLoS ONE

    Article Title: Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    doi: 10.1371/journal.pone.0013571

    Figure Lengend Snippet: L-NAME improves BCG-induced proliferation. NIH-3T3 fibroblast were treated with BCG (3×10 6 CFU/ml), L-NAME (2 mM) or BCG and L-NAME in presence of LY 294002 and PD 98059 for 24 and 48 h respectively. Cell viability was evaluated by MTS. a: p

    Article Snippet: To evaluate whether the MAPK or PI3K pathways were involved in BCG induction, NIH-3T3 cells were treated with BCG plus 10 and 20 µM of LY 294002 (Millipore) for 24 h or 10 and 50 uM of PD 98059 (Millipore) for 48 h. To evaluate NOS inhibitor activity on fibroblast viability, similar experiments were carried out in the presence of different concentrations of L-NAME (N-nitro-L-arginine-methyl-ester, Sigma).

    Techniques:

    BCG induces fibroblast proliferation. (A) NIH-3T3 fibroblast were treated with different concentration of BCG for 24 h. Cells were counted or (B) cell viability was evaluated by a non-radioactive cell titter method (MTS), vs control: p

    Journal: PLoS ONE

    Article Title: Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    doi: 10.1371/journal.pone.0013571

    Figure Lengend Snippet: BCG induces fibroblast proliferation. (A) NIH-3T3 fibroblast were treated with different concentration of BCG for 24 h. Cells were counted or (B) cell viability was evaluated by a non-radioactive cell titter method (MTS), vs control: p

    Article Snippet: To evaluate whether the MAPK or PI3K pathways were involved in BCG induction, NIH-3T3 cells were treated with BCG plus 10 and 20 µM of LY 294002 (Millipore) for 24 h or 10 and 50 uM of PD 98059 (Millipore) for 48 h. To evaluate NOS inhibitor activity on fibroblast viability, similar experiments were carried out in the presence of different concentrations of L-NAME (N-nitro-L-arginine-methyl-ester, Sigma).

    Techniques: Concentration Assay

    FGF-2 secreted by BCG-treated MACs induces NIH-3T3 cells proliferation. (A) Immunofluorescence staining with anti-FGF-2 antibody of RAW 264.7 MACs treated with BCG (3×10 6 CFU/ml) for 24 h. (B) Western blot from lysates of RAW 264.7 treated with BCG (3×10 6 CFU/ml) for 8 and 24 h. 20 ng of purified murine FGF-2 was used as a positive control. Relative expression level was normalized to GAPDH and referred as a fold change of control, a: p

    Journal: PLoS ONE

    Article Title: Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    doi: 10.1371/journal.pone.0013571

    Figure Lengend Snippet: FGF-2 secreted by BCG-treated MACs induces NIH-3T3 cells proliferation. (A) Immunofluorescence staining with anti-FGF-2 antibody of RAW 264.7 MACs treated with BCG (3×10 6 CFU/ml) for 24 h. (B) Western blot from lysates of RAW 264.7 treated with BCG (3×10 6 CFU/ml) for 8 and 24 h. 20 ng of purified murine FGF-2 was used as a positive control. Relative expression level was normalized to GAPDH and referred as a fold change of control, a: p

    Article Snippet: To evaluate whether the MAPK or PI3K pathways were involved in BCG induction, NIH-3T3 cells were treated with BCG plus 10 and 20 µM of LY 294002 (Millipore) for 24 h or 10 and 50 uM of PD 98059 (Millipore) for 48 h. To evaluate NOS inhibitor activity on fibroblast viability, similar experiments were carried out in the presence of different concentrations of L-NAME (N-nitro-L-arginine-methyl-ester, Sigma).

    Techniques: Magnetic Cell Separation, Immunofluorescence, Staining, Western Blot, Purification, Positive Control, Expressing

    BCG induces fibroblast differentiation. (A) Immunofluoresce staining of NIH-3T3 treated with BCG (3×10 6 CFU/ml) for 24 h revealed with anti-collagen I and anti-alpha-SMA antibody. Scale: bar = 100 um. (B) Western Blot from fibroblast homogenates treated with BCG (3×10 6 CFU/ml) at different times to determinate collagen I and alpha-SMA induction. (C) Densitometric units of collagen I or (D) alpha-SMA were determined using analysis software, relativized to beta-actin and referred as a fold change of control a: p

    Journal: PLoS ONE

    Article Title: Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    doi: 10.1371/journal.pone.0013571

    Figure Lengend Snippet: BCG induces fibroblast differentiation. (A) Immunofluoresce staining of NIH-3T3 treated with BCG (3×10 6 CFU/ml) for 24 h revealed with anti-collagen I and anti-alpha-SMA antibody. Scale: bar = 100 um. (B) Western Blot from fibroblast homogenates treated with BCG (3×10 6 CFU/ml) at different times to determinate collagen I and alpha-SMA induction. (C) Densitometric units of collagen I or (D) alpha-SMA were determined using analysis software, relativized to beta-actin and referred as a fold change of control a: p

    Article Snippet: To evaluate whether the MAPK or PI3K pathways were involved in BCG induction, NIH-3T3 cells were treated with BCG plus 10 and 20 µM of LY 294002 (Millipore) for 24 h or 10 and 50 uM of PD 98059 (Millipore) for 48 h. To evaluate NOS inhibitor activity on fibroblast viability, similar experiments were carried out in the presence of different concentrations of L-NAME (N-nitro-L-arginine-methyl-ester, Sigma).

    Techniques: Staining, Western Blot, Software

    ). The heavy arrows indicate methyltransferase open reading frames. Small arrows indicate primers used to amplify the 5′ and 3′ regions of mma3 . Thin solid lines represent the PCR products. Open squares, Sal I sites. Shown below the genomic regions are the amplicons from wild-type and mutant mma3 5′ regions, with Sal I sites indicated (note the additional site in the mutant mma3 ). Scale marker, 1,000 bp. (B) Purified 562-bp PCR products containing the 5′ end of the mma3 gene from the 13 BCG strains were digested with Sal I. Without digestion, a 562-bp product is seen. After incubation, the wild-type strains have three fragments of 303, 144, and 115 bp, while the mutant strains have four fragments of 246, 144, 115, and 57 bp. (C) TLC analysis of purified mycolic acids from various BCG substrains. The same BCG strains shown in the PCR-RFLP gel were analyzed by TLC for production of mycolic acids as described in Materials and Methods. The three bands obtained represent (from bottom to top) ketomycolates, methoxymycolates, and α-mycolates. It is seen in this figure that eight BCG strains lack methoxymycolates. These are the same strains that gave the extra Sal I band; they represent strains of BCG obtained from the Pasteur Institute in 1931 or later.

    Journal: Journal of Bacteriology

    Article Title: A Point Mutation in the mma3 Gene Is Responsible for Impaired Methoxymycolic Acid Production in Mycobacterium bovis BCG Strains Obtained after 1927

    doi:

    Figure Lengend Snippet: ). The heavy arrows indicate methyltransferase open reading frames. Small arrows indicate primers used to amplify the 5′ and 3′ regions of mma3 . Thin solid lines represent the PCR products. Open squares, Sal I sites. Shown below the genomic regions are the amplicons from wild-type and mutant mma3 5′ regions, with Sal I sites indicated (note the additional site in the mutant mma3 ). Scale marker, 1,000 bp. (B) Purified 562-bp PCR products containing the 5′ end of the mma3 gene from the 13 BCG strains were digested with Sal I. Without digestion, a 562-bp product is seen. After incubation, the wild-type strains have three fragments of 303, 144, and 115 bp, while the mutant strains have four fragments of 246, 144, 115, and 57 bp. (C) TLC analysis of purified mycolic acids from various BCG substrains. The same BCG strains shown in the PCR-RFLP gel were analyzed by TLC for production of mycolic acids as described in Materials and Methods. The three bands obtained represent (from bottom to top) ketomycolates, methoxymycolates, and α-mycolates. It is seen in this figure that eight BCG strains lack methoxymycolates. These are the same strains that gave the extra Sal I band; they represent strains of BCG obtained from the Pasteur Institute in 1931 or later.

    Article Snippet: These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.

    Techniques: Polymerase Chain Reaction, Mutagenesis, Marker, Purification, Incubation, Thin Layer Chromatography