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  • 99
    ATCC m bovis 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
    M Bovis Bcg, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche m bovis 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
    M Bovis Bcg, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Glaxo Smith m bovis bcg
    Antigen recognition by CD8 + T cells from <t>BCG-vaccinated</t> donors and TB patients. PBMC from 10 healthy donors (A) and 10 TB patients (B) were incubated in the presence of either M. <t>bovis</t> BCG (open bars) or M. tuberculosis H37Ra (solid bars) for 7 days. CD8 + T cells were purified by MACS. Autologus monocyte-derived macrophages were infected overnight with either M. bovis BCG, M. tuberculosis H37Ra, or rVV-expressing mycobacterial antigens. Effector cells were incubated with autologous, antigen-pulsed macrophages for 6 h at a 30:1 E/T ratio. CTL activity was measured as reflected by specific 51 Cr release. The results are expressed as the mean percent specific lysis ± the standard deviation.
    M Bovis Bcg, supplied by Glaxo Smith, used in various techniques. Bioz Stars score: 90/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC m bovis bcg frappier
    Analysis of trans -dominant mutants by gel electrophoresis. (a) Peroxidase activity gel of katG mutant plasmids in M. <t>bovis</t> <t>BCG</t> strain <t>ATCC</t> 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.
    M Bovis Bcg Frappier, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m bovis bcg pasteur
    High-throughput screening of the chemical compound library on hypoxic M. <t>bovis</t> <t>BCG</t> 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
    M Bovis Bcg Pasteur, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m bovis bcg tokyo
    Skin test reactivity of multiantigen cocktails in guinea pigs immunized with M. <t>bovis</t> <t>BCG</t> (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.
    M Bovis Bcg Tokyo, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Pasteur Institute m bovis bcg pasteur
    Growth of M. <t>bovis</t> <t>BCG</t> is inhibited by ADEP2, but FtsZ is not degraded A. In vitro degradation of FtsZ from M. bovis BCG (MTB FtsZ) or B. subtilis 168 (BS FtsZ) with ClpP proteins from M. tuberculosis or B. subtilis in the absence or presence of ADEP2 and ADEP4. B. Lysates of M. bovis BCG wildtype (wt), grown for 10–12 days in the presence of rising ADEP2 concentrations. SDS page (left), Western blot using an anti-MTB FtsZ antibody (right). C. Growth curve of M. bovis BCG wt in the absence or presence of ADEP2. D. Phase contrast images of M. bovis BCG wt without ADEP (upper lane) or with 16 μg ml −1 ADEP2 (lower lane) at distinct points in time.
    M Bovis Bcg Pasteur, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals m bovis bcg m bovis bcg
    Growth of M. <t>bovis</t> <t>BCG</t> is inhibited by ADEP2, but FtsZ is not degraded A. In vitro degradation of FtsZ from M. bovis BCG (MTB FtsZ) or B. subtilis 168 (BS FtsZ) with ClpP proteins from M. tuberculosis or B. subtilis in the absence or presence of ADEP2 and ADEP4. B. Lysates of M. bovis BCG wildtype (wt), grown for 10–12 days in the presence of rising ADEP2 concentrations. SDS page (left), Western blot using an anti-MTB FtsZ antibody (right). C. Growth curve of M. bovis BCG wt in the absence or presence of ADEP2. D. Phase contrast images of M. bovis BCG wt without ADEP (upper lane) or with 16 μg ml −1 ADEP2 (lower lane) at distinct points in time.
    M Bovis Bcg M Bovis Bcg, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m bovis bcg connaught
    M. <t>bovis</t> <t>BCG</t> 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).
    M Bovis Bcg Connaught, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m bovis bcg russia
    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. <t>bovis</t> <t>BCG-Russia.</t> 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 .
    M Bovis Bcg Russia, 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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    91
    BEI Resources m bovis bcg m bovis bcg
    Differential effects of transgenerational stress on acquired and innate immune responses at the F2 generation. F2 rats were vaccinated with 1 × 10 6 CFU of M. <t>bovis</t> <t>BCG</t> subcutaneously, euthanized 8 weeks later, and splenocytes cultured with M. bovis culture filtrate protein (CFP) to assess antigen-specific T cell responses or with Concanavalin-A as a strong non-specific stimulant. The cultures from F2 CSS females produced less IFN-γ in response to BCG-specific stimulation and to non-specific stimulation with Concanavalin-A than F2 rats with no exposure to CSS. In contrast, a trend for increased IL-6 production was observed under identical conditions in the same cultures. There was no clear relationship between CSS exposure in the F2 generation and TNF production.
    M Bovis Bcg M Bovis Bcg, supplied by BEI Resources, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Pasteur Institute m bovis bcg m bovis bcg pasteur strain 1172 p2
    Differential effects of transgenerational stress on acquired and innate immune responses at the F2 generation. F2 rats were vaccinated with 1 × 10 6 CFU of M. <t>bovis</t> <t>BCG</t> subcutaneously, euthanized 8 weeks later, and splenocytes cultured with M. bovis culture filtrate protein (CFP) to assess antigen-specific T cell responses or with Concanavalin-A as a strong non-specific stimulant. The cultures from F2 CSS females produced less IFN-γ in response to BCG-specific stimulation and to non-specific stimulation with Concanavalin-A than F2 rats with no exposure to CSS. In contrast, a trend for increased IL-6 production was observed under identical conditions in the same cultures. There was no clear relationship between CSS exposure in the F2 generation and TNF production.
    M Bovis Bcg M Bovis Bcg Pasteur Strain 1172 P2, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC m bovis bcg atcc 35743
    Differential effects of transgenerational stress on acquired and innate immune responses at the F2 generation. F2 rats were vaccinated with 1 × 10 6 CFU of M. <t>bovis</t> <t>BCG</t> subcutaneously, euthanized 8 weeks later, and splenocytes cultured with M. bovis culture filtrate protein (CFP) to assess antigen-specific T cell responses or with Concanavalin-A as a strong non-specific stimulant. The cultures from F2 CSS females produced less IFN-γ in response to BCG-specific stimulation and to non-specific stimulation with Concanavalin-A than F2 rats with no exposure to CSS. In contrast, a trend for increased IL-6 production was observed under identical conditions in the same cultures. There was no clear relationship between CSS exposure in the F2 generation and TNF production.
    M Bovis Bcg Atcc 35743, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Pasteur Institute m bovis bcg pasteur 1173p2
    Figure 5. M. <t>bovis</t> <t>BCG-induced</t> Mir155 and Mir31 are requisite to activate WNT-SHH pathway and regulate autophagy. ( A and B ) Immunoblotting was performed to analyze the expression of PPP2R5A and p-GSK3B ( A ) or WNT-SHH pathway ( B ) on M. bovis BCG
    M Bovis Bcg Pasteur 1173p2, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m bovis bcg tice atcc 27289
    qPCR amplification curves and standard curves of the serial dilution using (a) M. tuberculosis H37Rv; (b) M. <t>bovis</t> <t>BCG</t> Pasteur ATCC 35734; (c) M. microti ATCC 19422; and (d) M. caprae ZH 22914 for high‐resolution melting (HRM) assay 2
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    91
    ATCC m bovis bcg atcc 35747
    qPCR amplification curves and standard curves of the serial dilution using (a) M. tuberculosis H37Rv; (b) M. <t>bovis</t> <t>BCG</t> Pasteur ATCC 35734; (c) M. microti ATCC 19422; and (d) M. caprae ZH 22914 for high‐resolution melting (HRM) assay 2
    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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    86
    Bio-Rad m bovis bcg
    In vivo effects of M. <t>bovis</t> <t>BCG</t> strains overexpressing MabA_T191A and MabA_T191D. A , alteration of mycobacterial growth. Electrocompetent M. bovis BCG 1173P2 cells were transformed with the empty pMK1 construct, the pMK1_ mabA_WT , pMK1_ mabA_T191A , or pMK1_
    M Bovis Bcg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Connaught Labs m bovis bcg
    Prior bacillus Calmette Guérin <t>(BCG)</t> infection inhibited ovalbumin (OVA)-specific immunoglobulin E (IgE) production. Data from two independent experiments are shown. C57BL/6 mice were infected intravenously with 1×10 5 (a) or 1×10 6 (b) colony-forming units (CFUs) of Mycobacterium <t>bovis</t> BCG or treated with phosphate-buffered saline (PBS) alone. Two weeks after BCG inoculation, mice were immunized intraperitoneally with 2 μg of OVA in 2 mg of Al(OH) 3 (alum) adjuvant. Mice were bled at day 10 postimmunization for analysis of serum IgE production. Allergen (OVA)-specific IgE levels in individual mice were determined by passive cutaneous anaphylaxis (PCA) using female Sprague-Dawley rats. Geometric mean titres (±SEM) of each group, plotted on a log 2 scale, are presented. *represents P
    M Bovis Bcg, supplied by Connaught Labs, used in various techniques. Bioz Stars score: 86/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals m bovis bcg
    TLC-autoradiography of M. <t>bovis</t> <t>BCG</t> FAMEs and MAMEs after NAS-21 and NAS-91 analogue treatment. (A) NAS-21 analogue 1 (0–100 μg ml −1 ) and (B) NAS-91 analogue 15 (0–100 μg ml −1 ) were titrated into the M. bovis BCG/pVV16 cultures at an OD 600 of 0.4 prior to labelling with 1 μCi (37 kBq) [1,2- 14 C]acetate ml −1 for 8 h. [ 14 C]FAMEs and [ 14 C]MAMEs were extracted and resolved by TLC. An equivalent aliquot (20 μl) of the resulting solution of FAMEs and MAMEs was subjected to TLC using silica gel plates (5735 silica gel 60F 254 ; Merck), developed in petroleum ether/acetone (95 : 5, v/v). Autoradiograms were produced by overnight exposure to Kodak X-Omat AR film to reveal 14 C-labelled FAMEs and MAMEs.
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    Millipore m bovis bcg
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    Pasteur Institute m bovis bcg
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    Agilent technologies m bovis bcg
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    MedImmune m bovis bcg
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
    M Bovis Bcg, supplied by MedImmune, 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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    Pfizer Inc m bovis bcg danish
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    Thermo Fisher m bovis bcg
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
    M Bovis Bcg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    StressMarq recombinant m bovis bcg hsp65
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    DSMZ rna isolation m bovis bcg
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    Connaught Labs m bovis bcg strains conn
    (A) Cell survival of Mycobacterium <t>bovis</t> bacillus Calmette–Guérin ( M. bovis <t>BCG)-infected</t> MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P
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    ATCC m bovis bcg strain atcc 35748
    Hierarchical clustering of the transcriptional responses of M. <t>bovis</t> <t>BCG</t> growing in the chemostat model of TB and macrophage-adapted M. tuberculosis (naïve, activated, and NOS 2 knockout macrophages) and also after hydrogen treatment and growth
    M Bovis Bcg Strain Atcc 35748, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m bovis bcg strain 19015
    miR-146a promotes the survival of M. <t>bovis</t> <t>BCG</t> in macrophages. RAW264.7 cells were transfected with miR-146a mimic ( a – c ) or inhibitor ( d – f ) for 24 h, followed by BCG infection. The relative expression levels of miR-146a were determined by real-time PCR ( a , d ). Phagocytosis of Texas Red-labeled BCG was detected by flow cytometry ( b , e ). Mycobacterial viability was determined by CFU assay, and the survival was expressed as a percentage of the control ( c , f ). Data are shown as mean ± s.e.m. of three independent experiments. *p
    M Bovis Bcg Strain 19015, 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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    86
    Glaxo Smith m bovis bcg glaxo
    RaaS binds to its upstream region. (A) Schematic representation of the raaS operon in M. tuberculosis . (B) Intergenic region between raaS and rv1220c , containing the predicted −10 and −35 promoter elements (boxed). The RaaS-binding site is shown in red; the predicted raaS start and bcg1280c stop codons are underlined. (C) Influence of efflux inhibitors on survival of M. <t>bovis</t> <t>BCG</t> in extended stationary phase. ***, ethambutol (Emb)-, reserpine (Res)-, or CCCP-treated cells survived significantly better ( P
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    Image Search Results


    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

    Effect of BBD on M. bovis BCG growth in cells and outside cells in bacterial culture medium. BBD can inhibit M.bovis BCG growth in A549 ( A ) or THP-1 cells ( B ) and outside host cells in bacterial culture medium 7H9 ( C ) and the inhibitory effects are in dose-dependent manner.

    Journal: Scientific Reports

    Article Title: Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice

    doi: 10.1038/srep32725

    Figure Lengend Snippet: Effect of BBD on M. bovis BCG growth in cells and outside cells in bacterial culture medium. BBD can inhibit M.bovis BCG growth in A549 ( A ) or THP-1 cells ( B ) and outside host cells in bacterial culture medium 7H9 ( C ) and the inhibitory effects are in dose-dependent manner.

    Article Snippet: M. Smegmatis (ATCC 700084/mc(2)155) strains, M. bovis BCG Danish strain (ATCC 35733) and M. tuberculosis strains were grown in Middlebrook 7H9 broth and on Middlebrook 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase-enriched Middlebrook (OADC) according to our previously reported protocols .

    Techniques:

    Antigen recognition by CD8 + T cells from BCG-vaccinated donors and TB patients. PBMC from 10 healthy donors (A) and 10 TB patients (B) were incubated in the presence of either M. bovis BCG (open bars) or M. tuberculosis H37Ra (solid bars) for 7 days. CD8 + T cells were purified by MACS. Autologus monocyte-derived macrophages were infected overnight with either M. bovis BCG, M. tuberculosis H37Ra, or rVV-expressing mycobacterial antigens. Effector cells were incubated with autologous, antigen-pulsed macrophages for 6 h at a 30:1 E/T ratio. CTL activity was measured as reflected by specific 51 Cr release. The results are expressed as the mean percent specific lysis ± the standard deviation.

    Journal: Infection and Immunity

    Article Title: Human CD8+ T Cells Specific for Mycobacterium tuberculosis Secreted Antigens in Tuberculosis Patients and Healthy BCG-Vaccinated Controls in The Gambia

    doi:

    Figure Lengend Snippet: Antigen recognition by CD8 + T cells from BCG-vaccinated donors and TB patients. PBMC from 10 healthy donors (A) and 10 TB patients (B) were incubated in the presence of either M. bovis BCG (open bars) or M. tuberculosis H37Ra (solid bars) for 7 days. CD8 + T cells were purified by MACS. Autologus monocyte-derived macrophages were infected overnight with either M. bovis BCG, M. tuberculosis H37Ra, or rVV-expressing mycobacterial antigens. Effector cells were incubated with autologous, antigen-pulsed macrophages for 6 h at a 30:1 E/T ratio. CTL activity was measured as reflected by specific 51 Cr release. The results are expressed as the mean percent specific lysis ± the standard deviation.

    Article Snippet: The CD8+ T-cell response to M. bovis BCG and M. tuberculosis in healthy donors has previously been demonstrated to be MHC-I restricted ( , , – ).

    Techniques: Incubation, Purification, Magnetic Cell Separation, Derivative Assay, Infection, Expressing, CTL Assay, Activity Assay, Lysis, Standard Deviation

    CD8 + T-cell activation and perforin production in response to M. bovis BCG and M. tuberculosis H37Ra. PBMC from 10 healthy BCG-vaccinated donors (solid bars) and 10 pulmonary TB patients (open bars) were incubated with M. bovis BCG, M. tuberculosis H37Ra, or medium alone (No Ag) for 7 days. CD25 expression (A) and perforin production (B) by CD8 + T cells were measured by two-color flow cytometry. The results are presented as the mean percentage ± the standard deviation of CD8 + T cells staining positive for the marker.

    Journal: Infection and Immunity

    Article Title: Human CD8+ T Cells Specific for Mycobacterium tuberculosis Secreted Antigens in Tuberculosis Patients and Healthy BCG-Vaccinated Controls in The Gambia

    doi:

    Figure Lengend Snippet: CD8 + T-cell activation and perforin production in response to M. bovis BCG and M. tuberculosis H37Ra. PBMC from 10 healthy BCG-vaccinated donors (solid bars) and 10 pulmonary TB patients (open bars) were incubated with M. bovis BCG, M. tuberculosis H37Ra, or medium alone (No Ag) for 7 days. CD25 expression (A) and perforin production (B) by CD8 + T cells were measured by two-color flow cytometry. The results are presented as the mean percentage ± the standard deviation of CD8 + T cells staining positive for the marker.

    Article Snippet: The CD8+ T-cell response to M. bovis BCG and M. tuberculosis in healthy donors has previously been demonstrated to be MHC-I restricted ( , , – ).

    Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Cytometry, Standard Deviation, Staining, Marker

    MHC-I restriction of M. bovis BCG- and M. tuberculosis -specific CD8 + T cells from TB patients. 51 Cr-labeled macrophages were infected with either M. bovis BCG (A) or M. tuberculosis H37Ra (B) and preincubated for 1 h with w6/32 (anti-HLA-A, -B, or -C) or immunoglobulin G2a MAb as an isotype control; uninfected macrophages were also included as target cells (No Ag). CD8 + T cells prestimulated with either M. bovis BCG (open bars) or M. tuberculosis H37Ra (solid bars) for 7 days were purified by MACS and added at a 30:1 E/T ratio for the cytotoxicity assay. The data represent the mean specific lysis ± the standard deviation of 10 TB patients.

    Journal: Infection and Immunity

    Article Title: Human CD8+ T Cells Specific for Mycobacterium tuberculosis Secreted Antigens in Tuberculosis Patients and Healthy BCG-Vaccinated Controls in The Gambia

    doi:

    Figure Lengend Snippet: MHC-I restriction of M. bovis BCG- and M. tuberculosis -specific CD8 + T cells from TB patients. 51 Cr-labeled macrophages were infected with either M. bovis BCG (A) or M. tuberculosis H37Ra (B) and preincubated for 1 h with w6/32 (anti-HLA-A, -B, or -C) or immunoglobulin G2a MAb as an isotype control; uninfected macrophages were also included as target cells (No Ag). CD8 + T cells prestimulated with either M. bovis BCG (open bars) or M. tuberculosis H37Ra (solid bars) for 7 days were purified by MACS and added at a 30:1 E/T ratio for the cytotoxicity assay. The data represent the mean specific lysis ± the standard deviation of 10 TB patients.

    Article Snippet: The CD8+ T-cell response to M. bovis BCG and M. tuberculosis in healthy donors has previously been demonstrated to be MHC-I restricted ( , , – ).

    Techniques: Labeling, Infection, Purification, Magnetic Cell Separation, Cytotoxicity Assay, Lysis, Standard Deviation

    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

    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

    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

    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

    Rufomycin (RUF) analogues. a MIC against M. tuberculosis .

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus

    doi: 10.1128/AAC.02204-18

    Figure Lengend Snippet: Rufomycin (RUF) analogues. a MIC against M. tuberculosis .

    Article Snippet: The MIC against M. bovis (ATCC 35734) was determined with the MABA protocol.

    Techniques:

    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

    Growth of M. bovis BCG is inhibited by ADEP2, but FtsZ is not degraded A. In vitro degradation of FtsZ from M. bovis BCG (MTB FtsZ) or B. subtilis 168 (BS FtsZ) with ClpP proteins from M. tuberculosis or B. subtilis in the absence or presence of ADEP2 and ADEP4. B. Lysates of M. bovis BCG wildtype (wt), grown for 10–12 days in the presence of rising ADEP2 concentrations. SDS page (left), Western blot using an anti-MTB FtsZ antibody (right). C. Growth curve of M. bovis BCG wt in the absence or presence of ADEP2. D. Phase contrast images of M. bovis BCG wt without ADEP (upper lane) or with 16 μg ml −1 ADEP2 (lower lane) at distinct points in time.

    Journal: Molecular microbiology

    Article Title: Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease

    doi: 10.1111/mmi.13362

    Figure Lengend Snippet: Growth of M. bovis BCG is inhibited by ADEP2, but FtsZ is not degraded A. In vitro degradation of FtsZ from M. bovis BCG (MTB FtsZ) or B. subtilis 168 (BS FtsZ) with ClpP proteins from M. tuberculosis or B. subtilis in the absence or presence of ADEP2 and ADEP4. B. Lysates of M. bovis BCG wildtype (wt), grown for 10–12 days in the presence of rising ADEP2 concentrations. SDS page (left), Western blot using an anti-MTB FtsZ antibody (right). C. Growth curve of M. bovis BCG wt in the absence or presence of ADEP2. D. Phase contrast images of M. bovis BCG wt without ADEP (upper lane) or with 16 μg ml −1 ADEP2 (lower lane) at distinct points in time.

    Article Snippet: For establishing regulated expression of the clpP1 gene, a synthetic gene cassette ( hyg -P myc1 -4x tetO ; M. Alber and R. Kalscheuer, unpublished results) comprising a hygromycin resistance gene and the P myc1 promoter from M. smegmatis engineered to contain four tetO operator sites (serving as the DNA binding sites for the cognate repressor protein TetR) was inserted immediately upstream of the clpP1 start codon in M. bovis BCG Pasteur.

    Techniques: In Vitro, SDS Page, Western Blot

    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

    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

    Differential effects of transgenerational stress on acquired and innate immune responses at the F2 generation. F2 rats were vaccinated with 1 × 10 6 CFU of M. bovis BCG subcutaneously, euthanized 8 weeks later, and splenocytes cultured with M. bovis culture filtrate protein (CFP) to assess antigen-specific T cell responses or with Concanavalin-A as a strong non-specific stimulant. The cultures from F2 CSS females produced less IFN-γ in response to BCG-specific stimulation and to non-specific stimulation with Concanavalin-A than F2 rats with no exposure to CSS. In contrast, a trend for increased IL-6 production was observed under identical conditions in the same cultures. There was no clear relationship between CSS exposure in the F2 generation and TNF production.

    Journal: Brain Sciences

    Article Title: Transgenerational Social Stress Alters Immune–Behavior Associations and the Response to Vaccination

    doi: 10.3390/brainsci7070089

    Figure Lengend Snippet: Differential effects of transgenerational stress on acquired and innate immune responses at the F2 generation. F2 rats were vaccinated with 1 × 10 6 CFU of M. bovis BCG subcutaneously, euthanized 8 weeks later, and splenocytes cultured with M. bovis culture filtrate protein (CFP) to assess antigen-specific T cell responses or with Concanavalin-A as a strong non-specific stimulant. The cultures from F2 CSS females produced less IFN-γ in response to BCG-specific stimulation and to non-specific stimulation with Concanavalin-A than F2 rats with no exposure to CSS. In contrast, a trend for increased IL-6 production was observed under identical conditions in the same cultures. There was no clear relationship between CSS exposure in the F2 generation and TNF production.

    Article Snippet: Vaccination with M. Bovis BCG M. bovis BCG was obtained from BEI Resources and grown to mid-log phase in supplemented 7H9 broth as described [ ].

    Techniques: Cell Culture, Produced

    Figure 5. M. bovis BCG-induced Mir155 and Mir31 are requisite to activate WNT-SHH pathway and regulate autophagy. ( A and B ) Immunoblotting was performed to analyze the expression of PPP2R5A and p-GSK3B ( A ) or WNT-SHH pathway ( B ) on M. bovis BCG

    Journal: Autophagy

    Article Title: Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

    doi: 10.4161/auto.27225

    Figure Lengend Snippet: Figure 5. M. bovis BCG-induced Mir155 and Mir31 are requisite to activate WNT-SHH pathway and regulate autophagy. ( A and B ) Immunoblotting was performed to analyze the expression of PPP2R5A and p-GSK3B ( A ) or WNT-SHH pathway ( B ) on M. bovis BCG

    Article Snippet: M. bovis BCG Pasteur 1173P2 was obtained from Pasteur Institute, Paris, France and S. aureus, K. pneumoniae, S. flexneri, L. monocytogenes , and E. coli were obtained from The Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.

    Techniques: Expressing

    Figure 7. ALOXs dampen IFNG signaling to inhibit autophagy. ( A–C ) Knockdown of Alox5 and Alox15 was achieved by transfecting specific siRNAs in RAW 264.7 macrophages. 48 h post transfection, macrophages were infected with M. bovis BCG

    Journal: Autophagy

    Article Title: Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

    doi: 10.4161/auto.27225

    Figure Lengend Snippet: Figure 7. ALOXs dampen IFNG signaling to inhibit autophagy. ( A–C ) Knockdown of Alox5 and Alox15 was achieved by transfecting specific siRNAs in RAW 264.7 macrophages. 48 h post transfection, macrophages were infected with M. bovis BCG

    Article Snippet: M. bovis BCG Pasteur 1173P2 was obtained from Pasteur Institute, Paris, France and S. aureus, K. pneumoniae, S. flexneri, L. monocytogenes , and E. coli were obtained from The Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.

    Techniques: Transfection, Infection

    Figure 2. WNT-SHH signaling contributes to M. bovis BCG-mediated inhibition of autophagy. ( A ) Cotransfection of RAW 264.7 macrophages with pEGFP-MAP1LC3 and WNT5A or SHH overexpression constructs were performed, treated with IFNG and number of

    Journal: Autophagy

    Article Title: Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

    doi: 10.4161/auto.27225

    Figure Lengend Snippet: Figure 2. WNT-SHH signaling contributes to M. bovis BCG-mediated inhibition of autophagy. ( A ) Cotransfection of RAW 264.7 macrophages with pEGFP-MAP1LC3 and WNT5A or SHH overexpression constructs were performed, treated with IFNG and number of

    Article Snippet: M. bovis BCG Pasteur 1173P2 was obtained from Pasteur Institute, Paris, France and S. aureus, K. pneumoniae, S. flexneri, L. monocytogenes , and E. coli were obtained from The Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.

    Techniques: Inhibition, Cotransfection, Over Expression, Construct

    Figure 3. MTOR-PP2A regulate GSK3B activity to modulate autophagy. ( A and B ) Quantitative real-time RT-PCR ( A ) and immunoblot analysis ( B ) for Ppp2r5a and PPP2R5A, p-GSK3B respectively from M. bovis BCG-infected murine RAW 264.7 macrophages transfected

    Journal: Autophagy

    Article Title: Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

    doi: 10.4161/auto.27225

    Figure Lengend Snippet: Figure 3. MTOR-PP2A regulate GSK3B activity to modulate autophagy. ( A and B ) Quantitative real-time RT-PCR ( A ) and immunoblot analysis ( B ) for Ppp2r5a and PPP2R5A, p-GSK3B respectively from M. bovis BCG-infected murine RAW 264.7 macrophages transfected

    Article Snippet: M. bovis BCG Pasteur 1173P2 was obtained from Pasteur Institute, Paris, France and S. aureus, K. pneumoniae, S. flexneri, L. monocytogenes , and E. coli were obtained from The Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India.

    Techniques: Activity Assay, Quantitative RT-PCR, Infection, Transfection

    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

    In vivo effects of M. bovis BCG strains overexpressing MabA_T191A and MabA_T191D. A , alteration of mycobacterial growth. Electrocompetent M. bovis BCG 1173P2 cells were transformed with the empty pMK1 construct, the pMK1_ mabA_WT , pMK1_ mabA_T191A , or pMK1_

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of the Mycobacterium tuberculosis ?-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis *

    doi: 10.1074/jbc.M110.105189

    Figure Lengend Snippet: In vivo effects of M. bovis BCG strains overexpressing MabA_T191A and MabA_T191D. A , alteration of mycobacterial growth. Electrocompetent M. bovis BCG 1173P2 cells were transformed with the empty pMK1 construct, the pMK1_ mabA_WT , pMK1_ mabA_T191A , or pMK1_

    Article Snippet: LC-MS/MS identified an 80-Da mass increase corresponding to the peptide-(174–197), ANVTANVVAPGYIDTDM T RALDER, supporting the conclusion that MabA is phosphorylated in vivo in M. bovis BCG.

    Techniques: In Vivo, Transformation Assay, Construct

    Prior bacillus Calmette Guérin (BCG) infection inhibited ovalbumin (OVA)-specific immunoglobulin E (IgE) production. Data from two independent experiments are shown. C57BL/6 mice were infected intravenously with 1×10 5 (a) or 1×10 6 (b) colony-forming units (CFUs) of Mycobacterium bovis BCG or treated with phosphate-buffered saline (PBS) alone. Two weeks after BCG inoculation, mice were immunized intraperitoneally with 2 μg of OVA in 2 mg of Al(OH) 3 (alum) adjuvant. Mice were bled at day 10 postimmunization for analysis of serum IgE production. Allergen (OVA)-specific IgE levels in individual mice were determined by passive cutaneous anaphylaxis (PCA) using female Sprague-Dawley rats. Geometric mean titres (±SEM) of each group, plotted on a log 2 scale, are presented. *represents P

    Journal: Immunology

    Article Title: Systemic mycobacterial infection inhibits antigen-specific immunoglobulin E production, bronchial mucus production and eosinophilic inflammation induced by allergen

    doi: 10.1046/j.1365-2567.1999.00856.x

    Figure Lengend Snippet: Prior bacillus Calmette Guérin (BCG) infection inhibited ovalbumin (OVA)-specific immunoglobulin E (IgE) production. Data from two independent experiments are shown. C57BL/6 mice were infected intravenously with 1×10 5 (a) or 1×10 6 (b) colony-forming units (CFUs) of Mycobacterium bovis BCG or treated with phosphate-buffered saline (PBS) alone. Two weeks after BCG inoculation, mice were immunized intraperitoneally with 2 μg of OVA in 2 mg of Al(OH) 3 (alum) adjuvant. Mice were bled at day 10 postimmunization for analysis of serum IgE production. Allergen (OVA)-specific IgE levels in individual mice were determined by passive cutaneous anaphylaxis (PCA) using female Sprague-Dawley rats. Geometric mean titres (±SEM) of each group, plotted on a log 2 scale, are presented. *represents P

    Article Snippet: M. bovis BCG (BCG Vaccine; Connaught Laboratories Ltd, Willowdale, ON, Canada) was grown as dispersed cultures in Middlebrook’s 7H9 broth (Difco Laboratories Inc., Detroit, MI) containing 0·2% (v/v) glycerol and 0·05% (v/v) Tween-80 and supplemented with 10% (v/v) Middlebrook ADC enrichment (Difco).

    Techniques: Infection, Mouse Assay

    TLC-autoradiography of M. bovis BCG FAMEs and MAMEs after NAS-21 and NAS-91 analogue treatment. (A) NAS-21 analogue 1 (0–100 μg ml −1 ) and (B) NAS-91 analogue 15 (0–100 μg ml −1 ) were titrated into the M. bovis BCG/pVV16 cultures at an OD 600 of 0.4 prior to labelling with 1 μCi (37 kBq) [1,2- 14 C]acetate ml −1 for 8 h. [ 14 C]FAMEs and [ 14 C]MAMEs were extracted and resolved by TLC. An equivalent aliquot (20 μl) of the resulting solution of FAMEs and MAMEs was subjected to TLC using silica gel plates (5735 silica gel 60F 254 ; Merck), developed in petroleum ether/acetone (95 : 5, v/v). Autoradiograms were produced by overnight exposure to Kodak X-Omat AR film to reveal 14 C-labelled FAMEs and MAMEs.

    Journal: Microbiology

    Article Title: Synthesis and biological evaluation of NAS-21 and NAS-91 analogues as potential inhibitors of the mycobacterial FAS-II dehydratase enzyme Rv0636

    doi: 10.1099/mic.0.2008/017434-0

    Figure Lengend Snippet: TLC-autoradiography of M. bovis BCG FAMEs and MAMEs after NAS-21 and NAS-91 analogue treatment. (A) NAS-21 analogue 1 (0–100 μg ml −1 ) and (B) NAS-91 analogue 15 (0–100 μg ml −1 ) were titrated into the M. bovis BCG/pVV16 cultures at an OD 600 of 0.4 prior to labelling with 1 μCi (37 kBq) [1,2- 14 C]acetate ml −1 for 8 h. [ 14 C]FAMEs and [ 14 C]MAMEs were extracted and resolved by TLC. An equivalent aliquot (20 μl) of the resulting solution of FAMEs and MAMEs was subjected to TLC using silica gel plates (5735 silica gel 60F 254 ; Merck), developed in petroleum ether/acetone (95 : 5, v/v). Autoradiograms were produced by overnight exposure to Kodak X-Omat AR film to reveal 14 C-labelled FAMEs and MAMEs.

    Article Snippet: Liquid cultures of M. bovis BCG were grown at 37 °C in Sauton's medium containing 25 μg kanamycin ml−1 and 50 μg hygromycin ml−1 .

    Techniques: Thin Layer Chromatography, Autoradiography, Produced

    TLC-autoradiography of M. bovis BCG lipids after NAS-21 and NAS-91 analogue treatment. (A, D) Analysis of FAMEs and MAMEs following treatment with NAS-21 analogue 1 (20 μg ml −1 ) and NAS-91 analogue 15 (15 μg ml −1 ) and resolved by TLC using equal counts (50 000 c.p.m.) as described in Methods. Lipid extractions were performed as described by Dobson et al. (1985) and a 50 000 c.p.m. aliquot analysed using silica gel plates (5735 silica gel 60F 254 ; Merck). (B, E) Phenolic glycolipids (PGL) were identified by 2D TLC [direction 1, chloroform/methanol (94 : 4, v/v); direction 2, toluene/acetone (80 : 20, v/v)]. (C, F) Phospholipids (P), phosphatidylinositol (PI), acyl-phosphatidylinositol dimannoside (Ac 1 PIM 2 ) and diacyl-phosphatidylinositol dimannoside (Ac 2 PIM 2 ) were identified by 2D TLC [direction 1, chloroform/methanol/water (60 : 30 : 6, by vol.); direction 2, chloroform/acetic acid/methanol/water (40 : 25 : 3 : 6, by vol.)]. Autoradiograms were produced by overnight exposure to Kodak X-Omat AR film to reveal 14 C-labelled FAMEs, MAMEs and lipids.

    Journal: Microbiology

    Article Title: Synthesis and biological evaluation of NAS-21 and NAS-91 analogues as potential inhibitors of the mycobacterial FAS-II dehydratase enzyme Rv0636

    doi: 10.1099/mic.0.2008/017434-0

    Figure Lengend Snippet: TLC-autoradiography of M. bovis BCG lipids after NAS-21 and NAS-91 analogue treatment. (A, D) Analysis of FAMEs and MAMEs following treatment with NAS-21 analogue 1 (20 μg ml −1 ) and NAS-91 analogue 15 (15 μg ml −1 ) and resolved by TLC using equal counts (50 000 c.p.m.) as described in Methods. Lipid extractions were performed as described by Dobson et al. (1985) and a 50 000 c.p.m. aliquot analysed using silica gel plates (5735 silica gel 60F 254 ; Merck). (B, E) Phenolic glycolipids (PGL) were identified by 2D TLC [direction 1, chloroform/methanol (94 : 4, v/v); direction 2, toluene/acetone (80 : 20, v/v)]. (C, F) Phospholipids (P), phosphatidylinositol (PI), acyl-phosphatidylinositol dimannoside (Ac 1 PIM 2 ) and diacyl-phosphatidylinositol dimannoside (Ac 2 PIM 2 ) were identified by 2D TLC [direction 1, chloroform/methanol/water (60 : 30 : 6, by vol.); direction 2, chloroform/acetic acid/methanol/water (40 : 25 : 3 : 6, by vol.)]. Autoradiograms were produced by overnight exposure to Kodak X-Omat AR film to reveal 14 C-labelled FAMEs, MAMEs and lipids.

    Article Snippet: Liquid cultures of M. bovis BCG were grown at 37 °C in Sauton's medium containing 25 μg kanamycin ml−1 and 50 μg hygromycin ml−1 .

    Techniques: Thin Layer Chromatography, Autoradiography, Produced

    (A) Cell survival of Mycobacterium bovis bacillus Calmette–Guérin ( M. bovis BCG)-infected MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P

    Journal: Frontiers in Immunology

    Article Title: Functional Analysis of Genetic Variations in Surfactant Protein D in Mycobacterial Infection and Their Association With Tuberculosis

    doi: 10.3389/fimmu.2018.01543

    Figure Lengend Snippet: (A) Cell survival of Mycobacterium bovis bacillus Calmette–Guérin ( M. bovis BCG)-infected MH-S cells by native and four genetic variant recombinant SP-D (rSP-D) proteins measured using a Cell Counting Kit-8 assay. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] exhibited significantly better survival of infected MH-S cells than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P

    Article Snippet: To avoid effects on the viability of M. bovis BCG, penicillin and streptomycin were not added to the culture medium of the MH-S cells (Sigma-Aldrich, St. Louis, MO, USA) that were prepared for infection.

    Techniques: Infection, Variant Assay, Recombinant, Cell Counting

    (A) Dose-binding curves of native and four genetic variants of recombinant SP-D (rSP-D) proteins to Mycobacterium bovis bacillus Calmette–Guérin ( M. bovis BCG). rSP-D protein (1 and 10 µg/ml) were added to wells coated with ultraviolet-killed M. bovis BCG (10 6 bacteria), then quantitated using solid-phase bacterial ELISA. (B) Inhibition effects of maltose and EDTA on the binding activity of four variants of rSP-D to M. bovis BCG. Surfactant proteins D (SP-D) protein (1 µg/ml) in PBS with 2 mM CaCl 2 was added into M. bovis BCG-coated wells alone, or with 10 mM EDTA, or 100 mM maltose, or 100 mM glucose. The binding activity to M. bovis BCG of rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] was significantly higher than that of rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P

    Journal: Frontiers in Immunology

    Article Title: Functional Analysis of Genetic Variations in Surfactant Protein D in Mycobacterial Infection and Their Association With Tuberculosis

    doi: 10.3389/fimmu.2018.01543

    Figure Lengend Snippet: (A) Dose-binding curves of native and four genetic variants of recombinant SP-D (rSP-D) proteins to Mycobacterium bovis bacillus Calmette–Guérin ( M. bovis BCG). rSP-D protein (1 and 10 µg/ml) were added to wells coated with ultraviolet-killed M. bovis BCG (10 6 bacteria), then quantitated using solid-phase bacterial ELISA. (B) Inhibition effects of maltose and EDTA on the binding activity of four variants of rSP-D to M. bovis BCG. Surfactant proteins D (SP-D) protein (1 µg/ml) in PBS with 2 mM CaCl 2 was added into M. bovis BCG-coated wells alone, or with 10 mM EDTA, or 100 mM maltose, or 100 mM glucose. The binding activity to M. bovis BCG of rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] was significantly higher than that of rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P

    Article Snippet: To avoid effects on the viability of M. bovis BCG, penicillin and streptomycin were not added to the culture medium of the MH-S cells (Sigma-Aldrich, St. Louis, MO, USA) that were prepared for infection.

    Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Inhibition, Activity Assay

    (A) Inhibition of alveolar macrophage phagocytosis of Mycobacterium bovis bacillus Calmette–Guérin ( M. bovis BCG) by native and four genetic variants of recombinant SP-D (rSP-D) protein. Macrophages infected with FITC-labeled M. bovis BCG were analyzed using flow cytometry to assess the effects of the four rSP-D variants on the phagocytosis of MH-S cells. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] inhibited phagocytosis of M. bovis BCG by MH-S cells to a significantly greater extent than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P

    Journal: Frontiers in Immunology

    Article Title: Functional Analysis of Genetic Variations in Surfactant Protein D in Mycobacterial Infection and Their Association With Tuberculosis

    doi: 10.3389/fimmu.2018.01543

    Figure Lengend Snippet: (A) Inhibition of alveolar macrophage phagocytosis of Mycobacterium bovis bacillus Calmette–Guérin ( M. bovis BCG) by native and four genetic variants of recombinant SP-D (rSP-D) protein. Macrophages infected with FITC-labeled M. bovis BCG were analyzed using flow cytometry to assess the effects of the four rSP-D variants on the phagocytosis of MH-S cells. rSP-D with residue 11Thr [(92C/538A) and (92C/538G)] inhibited phagocytosis of M. bovis BCG by MH-S cells to a significantly greater extent than rSP-D with residue 11Met [(92T/538A) and (92T/538G)] (* P

    Article Snippet: To avoid effects on the viability of M. bovis BCG, penicillin and streptomycin were not added to the culture medium of the MH-S cells (Sigma-Aldrich, St. Louis, MO, USA) that were prepared for infection.

    Techniques: Inhibition, Recombinant, Infection, Labeling, Flow Cytometry, Cytometry

    Hierarchical clustering of the transcriptional responses of M. bovis BCG growing in the chemostat model of TB and macrophage-adapted M. tuberculosis (naïve, activated, and NOS 2 knockout macrophages) and also after hydrogen treatment and growth

    Journal:

    Article Title: Transcriptomic Analysis Identifies Growth Rate Modulation as a Component of the Adaptation of Mycobacteria to Survival inside the Macrophage ▿Transcriptomic Analysis Identifies Growth Rate Modulation as a Component of the Adaptation of Mycobacteria to Survival inside the Macrophage ▿ †

    doi: 10.1128/JB.01787-06

    Figure Lengend Snippet: Hierarchical clustering of the transcriptional responses of M. bovis BCG growing in the chemostat model of TB and macrophage-adapted M. tuberculosis (naïve, activated, and NOS 2 knockout macrophages) and also after hydrogen treatment and growth

    Article Snippet: M. bovis BCG strain ATCC 35748 was cultured in a 2-liter bioreactor (Adaptive Biosystem Voyager) under aerobic conditions at pH 6.6 as previously described ( ).

    Techniques: Knock-Out

    miR-146a promotes the survival of M. bovis BCG in macrophages. RAW264.7 cells were transfected with miR-146a mimic ( a – c ) or inhibitor ( d – f ) for 24 h, followed by BCG infection. The relative expression levels of miR-146a were determined by real-time PCR ( a , d ). Phagocytosis of Texas Red-labeled BCG was detected by flow cytometry ( b , e ). Mycobacterial viability was determined by CFU assay, and the survival was expressed as a percentage of the control ( c , f ). Data are shown as mean ± s.e.m. of three independent experiments. *p

    Journal: Scientific Reports

    Article Title: microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production

    doi: 10.1038/srep23351

    Figure Lengend Snippet: miR-146a promotes the survival of M. bovis BCG in macrophages. RAW264.7 cells were transfected with miR-146a mimic ( a – c ) or inhibitor ( d – f ) for 24 h, followed by BCG infection. The relative expression levels of miR-146a were determined by real-time PCR ( a , d ). Phagocytosis of Texas Red-labeled BCG was detected by flow cytometry ( b , e ). Mycobacterial viability was determined by CFU assay, and the survival was expressed as a percentage of the control ( c , f ). Data are shown as mean ± s.e.m. of three independent experiments. *p

    Article Snippet: M. bovis BCG strain 19015 was purchased from the American Type Culture Collection (ATCC), and were grown in Middlebrook 7H9 broth medium supplemented with 10% OADC and cultured in a standard culture incubator as reported before .

    Techniques: Transfection, Infection, Expressing, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cytometry, Colony-forming Unit Assay

    The expression of miR-146a were up-regulated after mycobacterial infection both in vivo and in vitro . Murine BMDMs ( a , b ) or RAW264.7 cells ( c , d ) were infected with M. bovis BCG at an MOI of 5 for the indicated time ( a , c ) or at the indicated MOIs for 24 h ( b , d ). Peritoneal lavage fluid and different organs were collected from BCG-infected or PBS-treated mice (n = 5).The expression levels of miR-146a were measured by real-time PCR ( e ). RAW264.7 cells were transfected with siRNA targeting MyD88 ( f ) or pretreated with IKKα/β inhibitor (BMS345541) ( g ), followed by BCG infection. The mRNA levels of MyD88 and miR-146a were measured by real-time PCR ( f , g ). Data are shown as mean ± s.e.m. of three independent experiments. *p

    Journal: Scientific Reports

    Article Title: microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production

    doi: 10.1038/srep23351

    Figure Lengend Snippet: The expression of miR-146a were up-regulated after mycobacterial infection both in vivo and in vitro . Murine BMDMs ( a , b ) or RAW264.7 cells ( c , d ) were infected with M. bovis BCG at an MOI of 5 for the indicated time ( a , c ) or at the indicated MOIs for 24 h ( b , d ). Peritoneal lavage fluid and different organs were collected from BCG-infected or PBS-treated mice (n = 5).The expression levels of miR-146a were measured by real-time PCR ( e ). RAW264.7 cells were transfected with siRNA targeting MyD88 ( f ) or pretreated with IKKα/β inhibitor (BMS345541) ( g ), followed by BCG infection. The mRNA levels of MyD88 and miR-146a were measured by real-time PCR ( f , g ). Data are shown as mean ± s.e.m. of three independent experiments. *p

    Article Snippet: M. bovis BCG strain 19015 was purchased from the American Type Culture Collection (ATCC), and were grown in Middlebrook 7H9 broth medium supplemented with 10% OADC and cultured in a standard culture incubator as reported before .

    Techniques: Expressing, Infection, In Vivo, In Vitro, Mouse Assay, Real-time Polymerase Chain Reaction, Transfection

    RaaS binds to its upstream region. (A) Schematic representation of the raaS operon in M. tuberculosis . (B) Intergenic region between raaS and rv1220c , containing the predicted −10 and −35 promoter elements (boxed). The RaaS-binding site is shown in red; the predicted raaS start and bcg1280c stop codons are underlined. (C) Influence of efflux inhibitors on survival of M. bovis BCG in extended stationary phase. ***, ethambutol (Emb)-, reserpine (Res)-, or CCCP-treated cells survived significantly better ( P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

    doi: 10.1128/AAC.02774-13

    Figure Lengend Snippet: RaaS binds to its upstream region. (A) Schematic representation of the raaS operon in M. tuberculosis . (B) Intergenic region between raaS and rv1220c , containing the predicted −10 and −35 promoter elements (boxed). The RaaS-binding site is shown in red; the predicted raaS start and bcg1280c stop codons are underlined. (C) Influence of efflux inhibitors on survival of M. bovis BCG in extended stationary phase. ***, ethambutol (Emb)-, reserpine (Res)-, or CCCP-treated cells survived significantly better ( P

    Article Snippet: In M. bovis BCG Glaxo and Pasteur strain, Bcg_1279c (which we designated RaaS, for r egulator of a ntimicrobial- a ssisted s urvival, based on the findings described below) only differs in its M. tuberculosis and M. bovis homologues by one amino acid (W113C), due to a single G-to-C substitution.

    Techniques: Binding Assay

    Survival of M. bovis BCG in prolonged stationary phase and the effect of antimicrobial treatment. (A) M. bovis BCG and M. tuberculosis were grown in sealed flasks to stationary phase and were incubated for a further 90 days. (B) One-month-old M. bovis BCG cultures were treated with antimicrobials and incubated for an additional 2 months before viability was assayed. **, statistically different from untreated control ( P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

    doi: 10.1128/AAC.02774-13

    Figure Lengend Snippet: Survival of M. bovis BCG in prolonged stationary phase and the effect of antimicrobial treatment. (A) M. bovis BCG and M. tuberculosis were grown in sealed flasks to stationary phase and were incubated for a further 90 days. (B) One-month-old M. bovis BCG cultures were treated with antimicrobials and incubated for an additional 2 months before viability was assayed. **, statistically different from untreated control ( P

    Article Snippet: In M. bovis BCG Glaxo and Pasteur strain, Bcg_1279c (which we designated RaaS, for r egulator of a ntimicrobial- a ssisted s urvival, based on the findings described below) only differs in its M. tuberculosis and M. bovis homologues by one amino acid (W113C), due to a single G-to-C substitution.

    Techniques: Incubation

    Effects of raaS overexpression or deletion on survival of M. bovis BCG in nonpermissive growth conditions in vitro and in vivo . (A) Strains containing pMind or pMind- raaS were incubated in tetracycline-supplemented medium for 107 days. **, statistically different survival between pMind- raaS and pMind ( P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

    doi: 10.1128/AAC.02774-13

    Figure Lengend Snippet: Effects of raaS overexpression or deletion on survival of M. bovis BCG in nonpermissive growth conditions in vitro and in vivo . (A) Strains containing pMind or pMind- raaS were incubated in tetracycline-supplemented medium for 107 days. **, statistically different survival between pMind- raaS and pMind ( P

    Article Snippet: In M. bovis BCG Glaxo and Pasteur strain, Bcg_1279c (which we designated RaaS, for r egulator of a ntimicrobial- a ssisted s urvival, based on the findings described below) only differs in its M. tuberculosis and M. bovis homologues by one amino acid (W113C), due to a single G-to-C substitution.

    Techniques: Over Expression, In Vitro, In Vivo, Incubation

    Interactions between amino acid residues in the wild type ( M. tuberculosis and M. bovis ) and those in the BCG CRP. (i) Leu47 residue of the wild-type CRP binding to residue Ile97; (ii) SNP Pro47 of BCG binding to Met104, showing the loss of one hydrogen

    Journal: Infection and Immunity

    Article Title: Single Nucleotide Polymorphisms That Cause Structural Changes in the Cyclic AMP Receptor Protein Transcriptional Regulator of the Tuberculosis Vaccine Strain Mycobacterium bovis BCG Alter Global Gene Expression without Attenuating Growth

    doi: 10.1128/IAI.01410-07

    Figure Lengend Snippet: Interactions between amino acid residues in the wild type ( M. tuberculosis and M. bovis ) and those in the BCG CRP. (i) Leu47 residue of the wild-type CRP binding to residue Ile97; (ii) SNP Pro47 of BCG binding to Met104, showing the loss of one hydrogen

    Article Snippet: The sequence of the protein (Fig. ) from all M. bovis BCG strains (e.g., Russia, Moreau, Birkhaug, Danish, Glaxo, Pasteur, and Tokyo) differs from the wild-type sequence (from M. bovis or M. tuberculosis ) in an amino acid residue in the HTH of the DNA-binding domain (Glu178Lys).

    Techniques: Binding Assay