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  • 90
    ATCC h37 rv rfpr
    H37 Rv Rfpr, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals h37 rv
    Exogenous vitamin B 12 represses transcription of M. tuberculosis metE . Reverse transcription (RT)-PCR analysis of metE expression in strains <t>H37Rv,</t> CDC1551, and the B 12 suppressor mutant B12P2 is shown. Cells were grown for 5 h in Sauton's minimal medium
    H37 Rv, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pasteur Institute m tuberculosis h37 rv
    Quantitative RT-PCR Expression levels of genes rv3614c , rv3865 , rv3867 , and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra:: phoP and <t>H37Rv</t> evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c , rv3865 , rv3867 , phoP , as well as of 16S rRNA are listed in Table S2 .
    M Tuberculosis H37 Rv, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals tuberculosis h37 rv
    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: <t>H37Rv</t> wildtype (234 cells), ΔponA1 (241 cells; approximate p-value
    Tuberculosis H37 Rv, supplied by MiddleBrook Pharmaceuticals, 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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    FUJIFILM mycobacterium tuberculosis strain h37 rv
    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: <t>H37Rv</t> wildtype (234 cells), ΔponA1 (241 cells; approximate p-value
    Mycobacterium Tuberculosis Strain H37 Rv, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources mycobacterium tuberculosis h37 rv
    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: <t>H37Rv</t> wildtype (234 cells), ΔponA1 (241 cells; approximate p-value
    Mycobacterium Tuberculosis H37 Rv, supplied by BEI Resources, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals mycobacterium tuberculosis h37 rv
    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: <t>H37Rv</t> wildtype (234 cells), ΔponA1 (241 cells; approximate p-value
    Mycobacterium Tuberculosis H37 Rv, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m tb h37 rv
    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: <t>H37Rv</t> wildtype (234 cells), ΔponA1 (241 cells; approximate p-value
    M Tb H37 Rv, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals mtb h37 rv middlebrook broth suspensions
    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: <t>H37Rv</t> wildtype (234 cells), ΔponA1 (241 cells; approximate p-value
    Mtb H37 Rv Middlebrook Broth Suspensions, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC m tuberculosis h37 rv
    rv3623/lpqG gene presence and transcription. ( A ) Amplification of hsp65 gene from gDNA isolated from: 1. <t>Mtb</t> <t>H37Rv;</t> 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: 50 bp molecular weight marker; ( B ) Amplification of rv3623 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control; ( C ) hsp65 gene amplification from cDNA of: 1. Mtb H37Rvplus synthesis; 2. Mtb H37Rvminus synthesis; 3. Mtb H37Raplus synthesis; 4. Mtb H37Raminus synthesis; 5. M. bovis plus synthesis; 6. M. bovis minus synthesis; 7. M. bovis BCGplus synthesis; 8. M. bovis BCGminus synthesis; 9. M. smegmatis plus synthesis; 10. M. smegmatis minus synthesis; 11. Positive PCR control ( Mtb H37RvgDNA); NC: Negative PCR control; MWM: 50 bp molecular weight marker; ( D ) rv3623 gene amplification from cDNA of: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: molecular weight marker.
    M Tuberculosis H37 Rv, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC mycobacterium tuberculosis h37 rv
    rv3623/lpqG gene presence and transcription. ( A ) Amplification of hsp65 gene from gDNA isolated from: 1. <t>Mtb</t> <t>H37Rv;</t> 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: 50 bp molecular weight marker; ( B ) Amplification of rv3623 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control; ( C ) hsp65 gene amplification from cDNA of: 1. Mtb H37Rvplus synthesis; 2. Mtb H37Rvminus synthesis; 3. Mtb H37Raplus synthesis; 4. Mtb H37Raminus synthesis; 5. M. bovis plus synthesis; 6. M. bovis minus synthesis; 7. M. bovis BCGplus synthesis; 8. M. bovis BCGminus synthesis; 9. M. smegmatis plus synthesis; 10. M. smegmatis minus synthesis; 11. Positive PCR control ( Mtb H37RvgDNA); NC: Negative PCR control; MWM: 50 bp molecular weight marker; ( D ) rv3623 gene amplification from cDNA of: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: molecular weight marker.
    Mycobacterium Tuberculosis H37 Rv, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC h37 rv isoniazid resistant
    rv3623/lpqG gene presence and transcription. ( A ) Amplification of hsp65 gene from gDNA isolated from: 1. <t>Mtb</t> <t>H37Rv;</t> 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: 50 bp molecular weight marker; ( B ) Amplification of rv3623 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control; ( C ) hsp65 gene amplification from cDNA of: 1. Mtb H37Rvplus synthesis; 2. Mtb H37Rvminus synthesis; 3. Mtb H37Raplus synthesis; 4. Mtb H37Raminus synthesis; 5. M. bovis plus synthesis; 6. M. bovis minus synthesis; 7. M. bovis BCGplus synthesis; 8. M. bovis BCGminus synthesis; 9. M. smegmatis plus synthesis; 10. M. smegmatis minus synthesis; 11. Positive PCR control ( Mtb H37RvgDNA); NC: Negative PCR control; MWM: 50 bp molecular weight marker; ( D ) rv3623 gene amplification from cDNA of: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: molecular weight marker.
    H37 Rv Isoniazid Resistant, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources h37rv
    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb <t>H37Rv</t> (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA
    H37rv, supplied by BEI Resources, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC h37 rv rifampicin resistant
    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb <t>H37Rv</t> (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA
    H37 Rv Rifampicin Resistant, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC h37 rv streptomycin resistant
    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb <t>H37Rv</t> (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA
    H37 Rv Streptomycin Resistant, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC drug susceptible h37 rv strain
    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb <t>H37Rv</t> (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA
    Drug Susceptible H37 Rv Strain, 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 etb resistant h37 rv atcc 35837
    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb <t>H37Rv</t> (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA
    Etb Resistant H37 Rv Atcc 35837, 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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    93
    BEI Resources mtb h37rv
    Bioenergetic analysis of <t>Mtb</t> <t>H37Rv,</t> cytochrome bd knockout ( cydKO ), and cydKO strain with Q203 resistance SNP (A317V) to ETC drugs. At indicated times, 2 g l −1 glucose was added, followed by either Q203, BDQ, or DCCD, followed by 2 μM CCCP. ( a ) 300 × MIC 50 BDQ treatment has a very similar effect on H37Rv and cydKO . ( b ) 300 × MIC 50 Q203 treatment causes H37Rv OCR to increase, however cydKO OCR drops to zero, while A317V OCR is unchanged. The untreated media OCR profiles of the Mtb H37Rv, cydKO and A317V strains are provided in Supplementary Fig. 4 . ( c ) 100 μM of the ATP synthase inhibitor DCCD causes a similar OCR increase to 30 × MIC 50 BDQ, although somewhat less in magnitude at these concentrations. ( d ) H37Rv Mtb was cultured for 24 h with 30 × MIC 50 of BDQ, Q203, CFZ, a RIF/INH combination, or media control. Relative abundance of ATP, ADP, and AMP was measured by LC-MS/MS. Standard deviations of three technical replicates are shown, all experiments were performed at least twice. * P
    Mtb H37rv, supplied by BEI Resources, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources tb h37rv
    Quinazoline derivatives target cytochrome bc 1 . (A) QcrA and QcrB of M . tb were modelled using the homology modelling webserver (Swissmodel) and homology based on chains A and M from the CryoEM structure of the III2IV2 respiratory supercomplex (PDB code 6HWH) from M . smegmatis . Clustering of mutations associated with resistance to quinazoline derivatives, AX-35, Q203, and LPZs occurs around the quinol oxidation site of QcrB and QcrA. (B) Cross-resistance of QuinR mutants to QcrB inhibitors. The MIC of the quinazoline derivatives, Q203, LPZs and AX-35 was determined and expressed as the fold-change of MIC of each compound compared to M . tb <t>H37Rv.</t> (C) Bioenergetic analysis of M . tb upon Q203 and 11626252 treatment followed by the uncoupler CCCP to stimulate maximum respiration. The oxygen consumption rates of wild-type H37Rv, H37Rv Δ cydAB , and H37Rv Δ cyd KO with a QcrB (A317V) mutation (Q203 resistance SNP) was monitored as described in the Material and methods section. (D) Intracellular ATP levels in H37Rv and quinazoline resistant strains were measured in the absence and presence of BDQ, Q203, and 11626252 at 2.5× MIC after 24 h using BacTiterGlo (Promega). Data from two independent experiments are presented as mean ± SD. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests (*, P
    Tb H37rv, supplied by BEI Resources, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Sequella h37rv
    phSGM2 accurately reported the STR MIC of a RIF r <t>H37Rv</t> variant. RIF r cells were added to the positive control and to STR- and RIF-containing wells of a Trek Sensititre dish and then infected with phSGM2. cSML generation was reduced to background levels
    H37rv, supplied by Sequella, used in various techniques. Bioz Stars score: 86/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Luminex h37rv
    Effect of infection in mice and rabbits with <t>H37Rv</t> and H37Rv::pPET1. (A) Growth of H37Rv and H37Rv::pPET1 in infected mouse lungs and spleen. Tissue homogenates from H37Rv::pPET1-infected mice were plated also on hygromycin-containing medium to determine
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    Glas-Col h37rv
    Histology of infected BALB/c mouse lungs using hematoxylin and eosin stain. BALB/c mice were infected by the low-dose aerosol method with the <t>H37Rv</t> strain of M. tuberculosis (A and B), the Δ lsr2 strain (C and D), or the Δ lsr2 complemented strain (E and F). Low-magnification photomicrographs (A, C, and E) or higher-magnification photomicrographs (B, D, and E) are shown.
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    Biotechnology Information h37rv
    Most recent common ancestors and deletion events during the evolution of the Mj sublineage of Nunavik. Because similar estimates with overlapping 95% highest posterior density intervals were obtained in all three MRCA analyses, MRCA dates obtained via analysis 1 are presented for simplicity. Arrows indicate the positions and annotations of the <t>H37Rv</t> reference genome. A phylogenetic cluster was defined as at least two genomes sharing a minimum of two single-nucleotide polymorphic loci. Gray and colored circles represent the common ancestors and phylogenetic clusters based on identified SNPs, respectively. Isolate 58385 had a unique single-nucleotide polymorphism profile (i.e., was not clustered) and, because the posterior density for the divergence date of this isolate was
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    ATCC antimycobacterial activity against mycobacterium tuberculosis h37 rv atcc 27194
    Most recent common ancestors and deletion events during the evolution of the Mj sublineage of Nunavik. Because similar estimates with overlapping 95% highest posterior density intervals were obtained in all three MRCA analyses, MRCA dates obtained via analysis 1 are presented for simplicity. Arrows indicate the positions and annotations of the <t>H37Rv</t> reference genome. A phylogenetic cluster was defined as at least two genomes sharing a minimum of two single-nucleotide polymorphic loci. Gray and colored circles represent the common ancestors and phylogenetic clusters based on identified SNPs, respectively. Isolate 58385 had a unique single-nucleotide polymorphism profile (i.e., was not clustered) and, because the posterior density for the divergence date of this isolate was
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    Millipore h37rv
    Live M. tuberculosis infection causes dendritic cell death and DNA fragmentation, without nuclear fragmentation . A - B . Dendritic cells (DCs) were infected, at MOI 10 with live/dead H37Ra or live/dead <t>H37Rv.</t> (U = uninfected, LH37Ra = live H37Ra, sH37Ra = streptomycin-killed H37Ra, LH37Rv = live H37Rv, iH37Rv = γ-irradiated H37Rv.) Cell death was measured by propidium iodide exclusion (A) 72 h post-infection or (B) 24 h post-infection on a GE IN Cell Analyzer 1000. (A - B) are means (± SEM) of 3 pooled donors. * p
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    Pasteur Institute h37rv
    Live M. tuberculosis infection causes dendritic cell death and DNA fragmentation, without nuclear fragmentation . A - B . Dendritic cells (DCs) were infected, at MOI 10 with live/dead H37Ra or live/dead <t>H37Rv.</t> (U = uninfected, LH37Ra = live H37Ra, sH37Ra = streptomycin-killed H37Ra, LH37Rv = live H37Rv, iH37Rv = γ-irradiated H37Rv.) Cell death was measured by propidium iodide exclusion (A) 72 h post-infection or (B) 24 h post-infection on a GE IN Cell Analyzer 1000. (A - B) are means (± SEM) of 3 pooled donors. * p
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    Promega h37rv
    Live M. tuberculosis infection causes dendritic cell death and DNA fragmentation, without nuclear fragmentation . A - B . Dendritic cells (DCs) were infected, at MOI 10 with live/dead H37Ra or live/dead <t>H37Rv.</t> (U = uninfected, LH37Ra = live H37Ra, sH37Ra = streptomycin-killed H37Ra, LH37Rv = live H37Rv, iH37Rv = γ-irradiated H37Rv.) Cell death was measured by propidium iodide exclusion (A) 72 h post-infection or (B) 24 h post-infection on a GE IN Cell Analyzer 1000. (A - B) are means (± SEM) of 3 pooled donors. * p
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    BEI Resources h37rv mtb wcl
    Humoral immune response in BAL and plasma after BCG immunization. <t>Mtb-responsive</t> antibody responses were assessed in BAL and plasma after BCG immunization. Mtb <t>WCL-specific</t> IgG, IgA and IgM antibody titres were measured from individual NHPs at various time points before and after BCG immunization. Shown are end-point titres for IgG and IgA and mid-point titres for IgM (in which the end point was not reached) a , Antibody titres in tenfold-concentrated BAL fluid (cohorts 1–4, n = 11–13 macaques except at weeks 2, 20 and 24, cohort 4 only, n = 3 macaques). In concentrated BAL fluid, antigen-responsive IgG, IgA and IgM were detected only in IV-BCG-immunized NHPs and returned to pre-vaccination levels by the time of challenge. b , Antibody titres in plasma ( n = 11–13 macaques). In plasma, both ID high and IV BCG elicited increased IgG and IgA antibody responses compared to ID low BCG. Data are geometric mean and s.d.; dashed line indicates assay limit of detection. A Kruskal–Wallis test was used to compare all vaccine groups to ID low at weeks 4, 16 and 24 (BAL) or weeks 4 and 24 (plasma); P values are from Dunn’s multiple comparison test (colour-coded to vaccine). Source Data
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    BEI Resources strain h37rv
    Hematopoietic miR-33 deficiency enhances autophagy gene expression in the lung and reduces Mtb burden in mice Analysis of ( a ) gene expression and ( b ) bacterial counts in the lungs of wild type (WT→WT) and Mir33 −/− ( Mir33 −/− →WT) bone marrow chimeric mice infected with <t>H37Rv</t> Mtb for the indicated time post-infection (p.i). * P ≤0.05, ** P ≤0.005 (Student’s t-test ( a , b )). Data are from 5 mice ( a, b ; mean ± s.e.m).
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    Pasteur Institute tuberculosis h37rv
    Hematopoietic miR-33 deficiency enhances autophagy gene expression in the lung and reduces Mtb burden in mice Analysis of ( a ) gene expression and ( b ) bacterial counts in the lungs of wild type (WT→WT) and Mir33 −/− ( Mir33 −/− →WT) bone marrow chimeric mice infected with <t>H37Rv</t> Mtb for the indicated time post-infection (p.i). * P ≤0.05, ** P ≤0.005 (Student’s t-test ( a , b )). Data are from 5 mice ( a, b ; mean ± s.e.m).
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    BEI Resources titrations m tb h37rv
    Hematopoietic miR-33 deficiency enhances autophagy gene expression in the lung and reduces Mtb burden in mice Analysis of ( a ) gene expression and ( b ) bacterial counts in the lungs of wild type (WT→WT) and Mir33 −/− ( Mir33 −/− →WT) bone marrow chimeric mice infected with <t>H37Rv</t> Mtb for the indicated time post-infection (p.i). * P ≤0.05, ** P ≤0.005 (Student’s t-test ( a , b )). Data are from 5 mice ( a, b ; mean ± s.e.m).
    Titrations M Tb H37rv, supplied by BEI Resources, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Exogenous vitamin B 12 represses transcription of M. tuberculosis metE . Reverse transcription (RT)-PCR analysis of metE expression in strains H37Rv, CDC1551, and the B 12 suppressor mutant B12P2 is shown. Cells were grown for 5 h in Sauton's minimal medium

    Journal:

    Article Title: A Riboswitch Regulates Expression of the Coenzyme B12-Independent Methionine Synthase in Mycobacterium tuberculosis: Implications for Differential Methionine Synthase Function in Strains H37Rv and CDC1551 ▿: Implications for Differential Methionine Synthase Function in Strains H37Rv and CDC1551 ▿ †

    doi: 10.1128/JB.00040-07

    Figure Lengend Snippet: Exogenous vitamin B 12 represses transcription of M. tuberculosis metE . Reverse transcription (RT)-PCR analysis of metE expression in strains H37Rv, CDC1551, and the B 12 suppressor mutant B12P2 is shown. Cells were grown for 5 h in Sauton's minimal medium

    Article Snippet: In both H37Rv and CDC1551, metE transcript levels were significantly reduced in B12 -supplemented medium, whereas expression of sigA was unaffected (Fig. ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis

    Quantitative RT-PCR Expression levels of genes rv3614c , rv3865 , rv3867 , and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra:: phoP and H37Rv evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c , rv3865 , rv3867 , phoP , as well as of 16S rRNA are listed in Table S2 .

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Quantitative RT-PCR Expression levels of genes rv3614c , rv3865 , rv3867 , and phoP in duplicate cultures of M. tuberculosis H37Ra and H37Ra:: phoP and H37Rv evaluated by quantitative RT-PCR (qRT-PCR). Genes rv3865 and rv3867 were included as controls as these genes are encoded in the region of difference 1 (RD1) and show similarity to rv3615/14c. The expression level of each gene in each strain is reported as the ratio between the expression level in M. tuberculosis H37Ra culture 1, used as reference. For each strain, values were normalized to the level of 16S rRNA. Note that in M. tuberculosis a single rrs gene is present. Primer and probe sequences of genes rv3614c , rv3865 , rv3867 , phoP , as well as of 16S rRNA are listed in Table S2 .

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: Quantitative RT-PCR, Expressing

    Secretion Analysis In vitro expression and secretion of ESAT-6 from M. tuberculosis H37Rv, H37Ra, MT103 phoP ko (SO2), and recombinant H37Ra complemented with integrating cosmids carrying genes phoP or rpsL from H37Rv. Total protein concentrations were determined by using Bio-Rad protein assay, and 15-μg samples were subjected to SDS-PAGE. Detection was carried out by using monoclonal anti-ESAT-6 antibody Hyb 76–8a.

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Secretion Analysis In vitro expression and secretion of ESAT-6 from M. tuberculosis H37Rv, H37Ra, MT103 phoP ko (SO2), and recombinant H37Ra complemented with integrating cosmids carrying genes phoP or rpsL from H37Rv. Total protein concentrations were determined by using Bio-Rad protein assay, and 15-μg samples were subjected to SDS-PAGE. Detection was carried out by using monoclonal anti-ESAT-6 antibody Hyb 76–8a.

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: In Vitro, Expressing, Recombinant, SDS Page

    Antigen-Specific IL-2 Production of T Cell Hybridomas Analysis of IL-2 secretion by anti-ESAT-6 or anti-Ag85A T cell hybridomas in response to recognition of antigen presented by bone marrow–derived dendritic cells (BM-DC) incubated with homologous or control peptides or infected with H37Rv, H37Ra or recombinant H37Ra strains.

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Antigen-Specific IL-2 Production of T Cell Hybridomas Analysis of IL-2 secretion by anti-ESAT-6 or anti-Ag85A T cell hybridomas in response to recognition of antigen presented by bone marrow–derived dendritic cells (BM-DC) incubated with homologous or control peptides or infected with H37Rv, H37Ra or recombinant H37Ra strains.

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: Derivative Assay, Incubation, Infection, Recombinant

    Macrophage Infection Studies Bone marrow–derived murine macrophages (BMM) were infected with M. tuberculosis H37Ra, H37Ra:: fadE5 , H37Ra:: phoP , and H37Rv at a MOI of ca. 1:1 and 10:1 bacteria per cell (A and B, respectively). The figure shows the means and standard deviations of CFU ratio values (CFU at days 1, 4, and 7 relative to CFU at 4 h) obtained in a representative experiment performed in quadruplicate. The significant difference levels in growth characteristics between H37Ra and other strains (H37Ra:: phoP and H37Rv) were determined by analysis of variance (ANOVA) (* p

    Journal: PLoS Pathogens

    Article Title: Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

    doi: 10.1371/journal.ppat.0040033

    Figure Lengend Snippet: Macrophage Infection Studies Bone marrow–derived murine macrophages (BMM) were infected with M. tuberculosis H37Ra, H37Ra:: fadE5 , H37Ra:: phoP , and H37Rv at a MOI of ca. 1:1 and 10:1 bacteria per cell (A and B, respectively). The figure shows the means and standard deviations of CFU ratio values (CFU at days 1, 4, and 7 relative to CFU at 4 h) obtained in a representative experiment performed in quadruplicate. The significant difference levels in growth characteristics between H37Ra and other strains (H37Ra:: phoP and H37Rv) were determined by analysis of variance (ANOVA) (* p

    Article Snippet: M. tuberculosis H37Ra corresponding to ATCC 25177 and M. tuberculosis H37Rv were taken from stocks held at the Pasteur Institute.

    Techniques: Infection, Derivative Assay

    PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: H37Rv wildtype (234 cells), ΔponA1 (241 cells; approximate p-value

    Journal: PLoS Pathogens

    Article Title: Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

    doi: 10.1371/journal.ppat.1005010

    Figure Lengend Snippet: PonA1’s glycan synthesis is required in M . smegmatis and catalytic activity promotes normal cell elongation. (A) Allelic exchange (see Fig 1A ) with vectors encoding alleles of PonA1 with varying catalytic activities (TG-, transglycosylase mutant; TP-, transpeptidase mutant; TG-TP-, transglycosylase and transpeptidase double mutant) demonstrates that a TG- allele fails to rescue bacterial survival. In contrast, a TP- allele complements bacterial growth. It is likely that PonA1’s synthesis of the glycan backbone of peptidoglycan is required for cell survival while other enzymes coordinate with PonA1 to crosslink those glycan strands into the existing sacculus. (B) M . tuberculosis strains were grown in culture and total cell length was measured: H37Rv wildtype (234 cells), ΔponA1 (241 cells; approximate p-value

    Article Snippet: M . tuberculosis H37Rv was cultured in Middlebrook 7H9 salts supplemented with OADC (oleic acid, albumin, dextrose, catalase [BD Biosciences, Franklin Lakes, NJ]), 0.25% glycerol, and 0.05% Tween-80 or plated on 7H10 agar.

    Techniques: Activity Assay, Mutagenesis

    PonA1 is essential in M . smegmatis and required for normal growth of M . tuberculosis . (A) An allelic exchange system in M . smegmatis provides an efficient method to test the importance of PonA1 for bacterial survival. PonA1 is essential in M . smegmatis , as allelic exchange with a vector encoding ponA1 complements bacterial growth, while allelic exchange with a negative control vector fails to rescue growth. (B) C57Bl6 mice were aerosol infected with H37Rv wildtype, ΔponA1 , ΔponA1 :: ponA1 wt , and ΔponA1 :: ponA1 TG- (an allele of PonA1 with a catalytic active site mutation in the transglycosylase (TG) domain), and CFU were enumerated were from lung and spleen homogenates at 15 and 42 days post infection (dpi). ΔponA1 and ΔponA1 :: ponA1 TG- cells are less fit than H37Rv wildtype or isogenic wildtype, respectively, at 15 and 42 dpi. Statistical significance was calculated by a one-tailed t-test (lungs at 15 dpi, H37Rv compared to ΔponA1 *** indicates p-value = 0.0005; lungs 42 dpi, H37Rv compared to ΔponA1 ** indicates p-value = 0.0089, and ΔponA1 :: ponA1 wt compared to ΔponA1 :: ponA1 TG- ** indicates p-value = 0.0042. Spleens at 42 dpi, H37Rv compared to ΔponA1 *** indicates p-value = 0.0001, and ΔponA1 :: ponA1 wt compared to ΔponA1 :: ponA1 TG- * indicates p-value = 0.0122). PonA1, while not required for growth of M . tuberculosis in culture, is required for normal bacterial multiplication and dissemination during infection in a mouse model of tuberculosis.

    Journal: PLoS Pathogens

    Article Title: Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

    doi: 10.1371/journal.ppat.1005010

    Figure Lengend Snippet: PonA1 is essential in M . smegmatis and required for normal growth of M . tuberculosis . (A) An allelic exchange system in M . smegmatis provides an efficient method to test the importance of PonA1 for bacterial survival. PonA1 is essential in M . smegmatis , as allelic exchange with a vector encoding ponA1 complements bacterial growth, while allelic exchange with a negative control vector fails to rescue growth. (B) C57Bl6 mice were aerosol infected with H37Rv wildtype, ΔponA1 , ΔponA1 :: ponA1 wt , and ΔponA1 :: ponA1 TG- (an allele of PonA1 with a catalytic active site mutation in the transglycosylase (TG) domain), and CFU were enumerated were from lung and spleen homogenates at 15 and 42 days post infection (dpi). ΔponA1 and ΔponA1 :: ponA1 TG- cells are less fit than H37Rv wildtype or isogenic wildtype, respectively, at 15 and 42 dpi. Statistical significance was calculated by a one-tailed t-test (lungs at 15 dpi, H37Rv compared to ΔponA1 *** indicates p-value = 0.0005; lungs 42 dpi, H37Rv compared to ΔponA1 ** indicates p-value = 0.0089, and ΔponA1 :: ponA1 wt compared to ΔponA1 :: ponA1 TG- ** indicates p-value = 0.0042. Spleens at 42 dpi, H37Rv compared to ΔponA1 *** indicates p-value = 0.0001, and ΔponA1 :: ponA1 wt compared to ΔponA1 :: ponA1 TG- * indicates p-value = 0.0122). PonA1, while not required for growth of M . tuberculosis in culture, is required for normal bacterial multiplication and dissemination during infection in a mouse model of tuberculosis.

    Article Snippet: M . tuberculosis H37Rv was cultured in Middlebrook 7H9 salts supplemented with OADC (oleic acid, albumin, dextrose, catalase [BD Biosciences, Franklin Lakes, NJ]), 0.25% glycerol, and 0.05% Tween-80 or plated on 7H10 agar.

    Techniques: Plasmid Preparation, Negative Control, Mouse Assay, Infection, Mutagenesis, One-tailed Test

    rv3623/lpqG gene presence and transcription. ( A ) Amplification of hsp65 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: 50 bp molecular weight marker; ( B ) Amplification of rv3623 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control; ( C ) hsp65 gene amplification from cDNA of: 1. Mtb H37Rvplus synthesis; 2. Mtb H37Rvminus synthesis; 3. Mtb H37Raplus synthesis; 4. Mtb H37Raminus synthesis; 5. M. bovis plus synthesis; 6. M. bovis minus synthesis; 7. M. bovis BCGplus synthesis; 8. M. bovis BCGminus synthesis; 9. M. smegmatis plus synthesis; 10. M. smegmatis minus synthesis; 11. Positive PCR control ( Mtb H37RvgDNA); NC: Negative PCR control; MWM: 50 bp molecular weight marker; ( D ) rv3623 gene amplification from cDNA of: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: molecular weight marker.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions

    doi: 10.3390/molecules23030526

    Figure Lengend Snippet: rv3623/lpqG gene presence and transcription. ( A ) Amplification of hsp65 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: 50 bp molecular weight marker; ( B ) Amplification of rv3623 gene from gDNA isolated from: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control; ( C ) hsp65 gene amplification from cDNA of: 1. Mtb H37Rvplus synthesis; 2. Mtb H37Rvminus synthesis; 3. Mtb H37Raplus synthesis; 4. Mtb H37Raminus synthesis; 5. M. bovis plus synthesis; 6. M. bovis minus synthesis; 7. M. bovis BCGplus synthesis; 8. M. bovis BCGminus synthesis; 9. M. smegmatis plus synthesis; 10. M. smegmatis minus synthesis; 11. Positive PCR control ( Mtb H37RvgDNA); NC: Negative PCR control; MWM: 50 bp molecular weight marker; ( D ) rv3623 gene amplification from cDNA of: 1. Mtb H37Rv; 2. Mtb H37Ra; 3. M. bovis ; 4. M. bovis BCG; 5. M. smegmatis ; 6. Positive PCR control; NC: Negative PCR control. MWM: molecular weight marker.

    Article Snippet: 4.2. lpqG gene Presence and Transcription The presence and transcription of the lpqG gene encoding the Rv3623 protein was determined in Mtb H37Rv H37Rv (ATCC 27294), Mtb H37Ra (ATCC 25177) and M. smegmatis (ATCC 19420) grown on Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC), for 20 or 5 days and M. bovis (ATCC 19210) and M. bovis BCG (ATCC 27291) on pyruvic acid-enriched culture medium, without glycerol, incubated at 37 °C.

    Techniques: Amplification, Isolation, Polymerase Chain Reaction, Molecular Weight, Marker

    Inhibiting entry to A549 cells and U937 cells. Inhibition assay regarding Mtb invasion of A549 and U937 cells; 2, 20 and 200 µM concentrations were used for both cell lines. Mtb H37Rv lysate at 200 µg/mL concentration was used as inhibition control (C+). Mycobacteria within A549 and U937 cells can be observed in the fluorescence microscopic image (100×).

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions

    doi: 10.3390/molecules23030526

    Figure Lengend Snippet: Inhibiting entry to A549 cells and U937 cells. Inhibition assay regarding Mtb invasion of A549 and U937 cells; 2, 20 and 200 µM concentrations were used for both cell lines. Mtb H37Rv lysate at 200 µg/mL concentration was used as inhibition control (C+). Mycobacteria within A549 and U937 cells can be observed in the fluorescence microscopic image (100×).

    Article Snippet: 4.2. lpqG gene Presence and Transcription The presence and transcription of the lpqG gene encoding the Rv3623 protein was determined in Mtb H37Rv H37Rv (ATCC 27294), Mtb H37Ra (ATCC 25177) and M. smegmatis (ATCC 19420) grown on Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC), for 20 or 5 days and M. bovis (ATCC 19210) and M. bovis BCG (ATCC 27291) on pyruvic acid-enriched culture medium, without glycerol, incubated at 37 °C.

    Techniques: Inhibition, Concentration Assay, Fluorescence

    Western blot recognition of Rv3623 in M. tuberculosis . Lane 1: pre-immune sera. Lane 2: post-third immunisation of the 16660/16665/BSA mixture against lysed Mtb H37Rv. Lanes 3 and 7: recognition of lysed Mtb H37Rv by hyper-immune sera control (obtained by inoculating mice with Mtb H37Rv sonicate). Identification of membrane (lane 4), wall (lane 5) and cytosol proteins (lane 6) lysed from H37Rv by sera from mice immunized with the 16660/16665/BSA mixture (25 µg antigen was added to each lane).

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions

    doi: 10.3390/molecules23030526

    Figure Lengend Snippet: Western blot recognition of Rv3623 in M. tuberculosis . Lane 1: pre-immune sera. Lane 2: post-third immunisation of the 16660/16665/BSA mixture against lysed Mtb H37Rv. Lanes 3 and 7: recognition of lysed Mtb H37Rv by hyper-immune sera control (obtained by inoculating mice with Mtb H37Rv sonicate). Identification of membrane (lane 4), wall (lane 5) and cytosol proteins (lane 6) lysed from H37Rv by sera from mice immunized with the 16660/16665/BSA mixture (25 µg antigen was added to each lane).

    Article Snippet: 4.2. lpqG gene Presence and Transcription The presence and transcription of the lpqG gene encoding the Rv3623 protein was determined in Mtb H37Rv H37Rv (ATCC 27294), Mtb H37Ra (ATCC 25177) and M. smegmatis (ATCC 19420) grown on Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC), for 20 or 5 days and M. bovis (ATCC 19210) and M. bovis BCG (ATCC 27291) on pyruvic acid-enriched culture medium, without glycerol, incubated at 37 °C.

    Techniques: Western Blot, Mouse Assay

    Antigen recognition of Rv3623’s peptides evaluated in sera from patients having active tuberculosis (ATB), latent tuberculosis (LTB) and healthy patients (HC). PBS was used as negative control and Mtb H37Rv lysate and Sauton medium supernatant proteins released as antigen were also used. Positive control involved using mouse polyclonal sera against peptides 16665 and 16660.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions

    doi: 10.3390/molecules23030526

    Figure Lengend Snippet: Antigen recognition of Rv3623’s peptides evaluated in sera from patients having active tuberculosis (ATB), latent tuberculosis (LTB) and healthy patients (HC). PBS was used as negative control and Mtb H37Rv lysate and Sauton medium supernatant proteins released as antigen were also used. Positive control involved using mouse polyclonal sera against peptides 16665 and 16660.

    Article Snippet: 4.2. lpqG gene Presence and Transcription The presence and transcription of the lpqG gene encoding the Rv3623 protein was determined in Mtb H37Rv H37Rv (ATCC 27294), Mtb H37Ra (ATCC 25177) and M. smegmatis (ATCC 19420) grown on Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC), for 20 or 5 days and M. bovis (ATCC 19210) and M. bovis BCG (ATCC 27291) on pyruvic acid-enriched culture medium, without glycerol, incubated at 37 °C.

    Techniques: Negative Control, Positive Control

    Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb H37Rv (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA

    Journal: Mucosal immunology

    Article Title: Novel role for IL-22 in protection during chronic Mycobacterium tuberculosis HN878 infection

    doi: 10.1038/mi.2017.15

    Figure Lengend Snippet: Infection with Mtb HN878 triggers increased IL-22 production through a TLR2-dependent pathway Lung cells isolated from B6, Il1r-/- and Tlr2-/- mice were infected with either Mtb H37Rv (Rv) or Mtb HN878 (HN) at MOI of 0.1 for 7 days, and the protein levels of ( a ) IL-1β and ( b ) IL-22 were assessed in culture supernatants by ELISA and luminex assay, respectively. Lung cells isolated from B6 mice were treated with cell wall extracted from Mtb H37Rv (Rv) or Mtb HN878 (HN), (10μg/ml each). Protein levels of ( c ) IL-1β and ( d ) IL-22 were assessed by ELISA and luminex assay respectively. PBMCs were isolated from ATB patients and were stimulated with Mtb H37Rv (Rv) or Mtb HN878 (HN) cell wall extracts (20 μg/ml each) and the protein levels of ( e ) IL-22 were measured by luminex assays. Error bars represent means ± s.e.m. n = 3-5 for a-d. n = 17 for human ATB patients for e. ND, not detected; UI, uninfected; UN, untreated. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 by ( a,b ) one-way ANOVA, ( c,d ) Student's t test ( e ) two-way ANOVA

    Article Snippet: H37Rv (NR14828) or HN878 (NR14830) cell wall preparations were commercially obtained from BEI Resources, under National Institutes of Health [NIH] contract AI-75320.

    Techniques: Infection, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Luminex

    Bioenergetic analysis of Mtb H37Rv, cytochrome bd knockout ( cydKO ), and cydKO strain with Q203 resistance SNP (A317V) to ETC drugs. At indicated times, 2 g l −1 glucose was added, followed by either Q203, BDQ, or DCCD, followed by 2 μM CCCP. ( a ) 300 × MIC 50 BDQ treatment has a very similar effect on H37Rv and cydKO . ( b ) 300 × MIC 50 Q203 treatment causes H37Rv OCR to increase, however cydKO OCR drops to zero, while A317V OCR is unchanged. The untreated media OCR profiles of the Mtb H37Rv, cydKO and A317V strains are provided in Supplementary Fig. 4 . ( c ) 100 μM of the ATP synthase inhibitor DCCD causes a similar OCR increase to 30 × MIC 50 BDQ, although somewhat less in magnitude at these concentrations. ( d ) H37Rv Mtb was cultured for 24 h with 30 × MIC 50 of BDQ, Q203, CFZ, a RIF/INH combination, or media control. Relative abundance of ATP, ADP, and AMP was measured by LC-MS/MS. Standard deviations of three technical replicates are shown, all experiments were performed at least twice. * P

    Journal: Nature Communications

    Article Title: Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

    doi: 10.1038/ncomms12393

    Figure Lengend Snippet: Bioenergetic analysis of Mtb H37Rv, cytochrome bd knockout ( cydKO ), and cydKO strain with Q203 resistance SNP (A317V) to ETC drugs. At indicated times, 2 g l −1 glucose was added, followed by either Q203, BDQ, or DCCD, followed by 2 μM CCCP. ( a ) 300 × MIC 50 BDQ treatment has a very similar effect on H37Rv and cydKO . ( b ) 300 × MIC 50 Q203 treatment causes H37Rv OCR to increase, however cydKO OCR drops to zero, while A317V OCR is unchanged. The untreated media OCR profiles of the Mtb H37Rv, cydKO and A317V strains are provided in Supplementary Fig. 4 . ( c ) 100 μM of the ATP synthase inhibitor DCCD causes a similar OCR increase to 30 × MIC 50 BDQ, although somewhat less in magnitude at these concentrations. ( d ) H37Rv Mtb was cultured for 24 h with 30 × MIC 50 of BDQ, Q203, CFZ, a RIF/INH combination, or media control. Relative abundance of ATP, ADP, and AMP was measured by LC-MS/MS. Standard deviations of three technical replicates are shown, all experiments were performed at least twice. * P

    Article Snippet: Mtb H37Rv was obtained from BEI Resources (NR-123).

    Techniques: Knock-Out, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Analysis of combination treatment of Mtb H37Rv. Indicated additions are 2 g l −1 glucose (Glc), drug combination, and 2 μM CCCP as an uncoupler to stimulate maximum respiration. OCR and ECAR are shown as a per cent of baseline values with standard deviation of three replicate experiments performed. Kill curves were constructed from CFUs measured over 20 days of treated cells grown in 7H9 with 2 g l −1 of glucose and 0.01% tyloxopol. Limit of detection (LOD) is 10 CFU per ml. All drugs were added at 30 × MIC 50 values. Two separate kill curve experiments were performed with the same results, representative data is shown. ( a ) BDQ, Q203, BDQ/Q203, and BDQ/Q203/CFZ combinations. ( b ) BDQ, CFZ, BDQ/CFZ and BDQ/Q203/CFZ combinations. ( c ) Q203, CFZ, Q203/CFZ, and BDQ/Q203/CFZ combinations.

    Journal: Nature Communications

    Article Title: Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

    doi: 10.1038/ncomms12393

    Figure Lengend Snippet: Analysis of combination treatment of Mtb H37Rv. Indicated additions are 2 g l −1 glucose (Glc), drug combination, and 2 μM CCCP as an uncoupler to stimulate maximum respiration. OCR and ECAR are shown as a per cent of baseline values with standard deviation of three replicate experiments performed. Kill curves were constructed from CFUs measured over 20 days of treated cells grown in 7H9 with 2 g l −1 of glucose and 0.01% tyloxopol. Limit of detection (LOD) is 10 CFU per ml. All drugs were added at 30 × MIC 50 values. Two separate kill curve experiments were performed with the same results, representative data is shown. ( a ) BDQ, Q203, BDQ/Q203, and BDQ/Q203/CFZ combinations. ( b ) BDQ, CFZ, BDQ/CFZ and BDQ/Q203/CFZ combinations. ( c ) Q203, CFZ, Q203/CFZ, and BDQ/Q203/CFZ combinations.

    Article Snippet: Mtb H37Rv was obtained from BEI Resources (NR-123).

    Techniques: Gas Chromatography, Standard Deviation, Construct

    Analysis of the effects of different Mtb ETC targeting drug combinations on mammalian cell lines. Cell lines were treated with the ETC targeting drugs at 30 × MIC 50 concentrations. Oligomycin was used at a concentration of 3.0 μM and 1.5 μM for HepG2 and RAW264.7 cells, respectively. FCCP was used at a concentration of 1.5 μM, and rotenone and antimycin A were both used at a concentration of 0.5 μM. HepG2 cells ( a – c ) and RAW264.7 cells ( d – f ) were seeded at a density of 25,000 and 65,000 cells per well, respectively. In both the HepG2 and RAW264.7 cell lines the Mtb ETC targeting drug combination had similar effects on the four bioenergetic parameters as observed in the controls. These parameters include basal respiration (BR), ATP turnover (AT, an indication of the amount of O 2 consumed to produce ATP in the mammalian ETC), spare respiratory capacity (SRC, an indication of the cell's capacity to respond under stressful conditions) and non-mitochondrial respiration (non-MR, e.g . NADPH oxidase mediated reduction of O 2 during the oxidative burst). Experiments were performed twice. Kill curves ( g – i ) were constructed by infecting 200,000 RAW264.7 cells per well with Mtb H37Rv at a MOI of 1 and drug treating at 30 × MIC 50 concentrations, for 4 days. CFU data is presented as the mean percentage±s.d. reduction in CFU from Day 0 of three independent experiments performed in triplicate.

    Journal: Nature Communications

    Article Title: Turning the respiratory flexibility of Mycobacterium tuberculosis against itself

    doi: 10.1038/ncomms12393

    Figure Lengend Snippet: Analysis of the effects of different Mtb ETC targeting drug combinations on mammalian cell lines. Cell lines were treated with the ETC targeting drugs at 30 × MIC 50 concentrations. Oligomycin was used at a concentration of 3.0 μM and 1.5 μM for HepG2 and RAW264.7 cells, respectively. FCCP was used at a concentration of 1.5 μM, and rotenone and antimycin A were both used at a concentration of 0.5 μM. HepG2 cells ( a – c ) and RAW264.7 cells ( d – f ) were seeded at a density of 25,000 and 65,000 cells per well, respectively. In both the HepG2 and RAW264.7 cell lines the Mtb ETC targeting drug combination had similar effects on the four bioenergetic parameters as observed in the controls. These parameters include basal respiration (BR), ATP turnover (AT, an indication of the amount of O 2 consumed to produce ATP in the mammalian ETC), spare respiratory capacity (SRC, an indication of the cell's capacity to respond under stressful conditions) and non-mitochondrial respiration (non-MR, e.g . NADPH oxidase mediated reduction of O 2 during the oxidative burst). Experiments were performed twice. Kill curves ( g – i ) were constructed by infecting 200,000 RAW264.7 cells per well with Mtb H37Rv at a MOI of 1 and drug treating at 30 × MIC 50 concentrations, for 4 days. CFU data is presented as the mean percentage±s.d. reduction in CFU from Day 0 of three independent experiments performed in triplicate.

    Article Snippet: Mtb H37Rv was obtained from BEI Resources (NR-123).

    Techniques: Concentration Assay, Construct

    In vitro differential mycobacterial control is improved among individuals with hookworm infection in whole blood and PBMC and is lost after helminth treatment. Whole blood and PBMC MGIT using M.tb H37Rv was performed to assess mycobacterial control. Whole blood samples were available from uninfected controls ( n = 9) and matched pre- and post-treatment samples from hookworm infected individuals ( n = 13) (A) . The relationship between mycobacterial net growth and hematology parameters was investigated. After testing for normality, Spearman's correlations were calculated between hematology data and all whole blood M.tb H37Rv MGIT results ( n = 34) and showed a significant negative correlation growth and eosinophil count (B) . MGIA was also performed using cryopreserved PBMC with matched pre- and post-treatment PBMC samples from individuals with hookworm infection ( n = 12) and controls ( n = 9) (C) . For MGIT data, points represent the mean of duplicates; bars represent mean values with SD. A one-way multiple comparison ANOVA with Tukey's post-test correction was performed between the groups. HW, hookworm; Rx, treatment; ns, non-significant; ** p

    Journal: Frontiers in Immunology

    Article Title: Human Hookworm Infection Enhances Mycobacterial Growth Inhibition and Associates With Reduced Risk of Tuberculosis Infection

    doi: 10.3389/fimmu.2018.02893

    Figure Lengend Snippet: In vitro differential mycobacterial control is improved among individuals with hookworm infection in whole blood and PBMC and is lost after helminth treatment. Whole blood and PBMC MGIT using M.tb H37Rv was performed to assess mycobacterial control. Whole blood samples were available from uninfected controls ( n = 9) and matched pre- and post-treatment samples from hookworm infected individuals ( n = 13) (A) . The relationship between mycobacterial net growth and hematology parameters was investigated. After testing for normality, Spearman's correlations were calculated between hematology data and all whole blood M.tb H37Rv MGIT results ( n = 34) and showed a significant negative correlation growth and eosinophil count (B) . MGIA was also performed using cryopreserved PBMC with matched pre- and post-treatment PBMC samples from individuals with hookworm infection ( n = 12) and controls ( n = 9) (C) . For MGIT data, points represent the mean of duplicates; bars represent mean values with SD. A one-way multiple comparison ANOVA with Tukey's post-test correction was performed between the groups. HW, hookworm; Rx, treatment; ns, non-significant; ** p

    Article Snippet: Microtiter plates were coated overnight at room temperature with a 1:1 mix of ESAT-6/CFP-10 (10 μg/ml each) prepared from M.tb H37Rv (BEI Resources).

    Techniques: In Vitro, Infection

    Quinazoline derivatives target cytochrome bc 1 . (A) QcrA and QcrB of M . tb were modelled using the homology modelling webserver (Swissmodel) and homology based on chains A and M from the CryoEM structure of the III2IV2 respiratory supercomplex (PDB code 6HWH) from M . smegmatis . Clustering of mutations associated with resistance to quinazoline derivatives, AX-35, Q203, and LPZs occurs around the quinol oxidation site of QcrB and QcrA. (B) Cross-resistance of QuinR mutants to QcrB inhibitors. The MIC of the quinazoline derivatives, Q203, LPZs and AX-35 was determined and expressed as the fold-change of MIC of each compound compared to M . tb H37Rv. (C) Bioenergetic analysis of M . tb upon Q203 and 11626252 treatment followed by the uncoupler CCCP to stimulate maximum respiration. The oxygen consumption rates of wild-type H37Rv, H37Rv Δ cydAB , and H37Rv Δ cyd KO with a QcrB (A317V) mutation (Q203 resistance SNP) was monitored as described in the Material and methods section. (D) Intracellular ATP levels in H37Rv and quinazoline resistant strains were measured in the absence and presence of BDQ, Q203, and 11626252 at 2.5× MIC after 24 h using BacTiterGlo (Promega). Data from two independent experiments are presented as mean ± SD. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests (*, P

    Journal: PLoS Pathogens

    Article Title: New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis

    doi: 10.1371/journal.ppat.1008270

    Figure Lengend Snippet: Quinazoline derivatives target cytochrome bc 1 . (A) QcrA and QcrB of M . tb were modelled using the homology modelling webserver (Swissmodel) and homology based on chains A and M from the CryoEM structure of the III2IV2 respiratory supercomplex (PDB code 6HWH) from M . smegmatis . Clustering of mutations associated with resistance to quinazoline derivatives, AX-35, Q203, and LPZs occurs around the quinol oxidation site of QcrB and QcrA. (B) Cross-resistance of QuinR mutants to QcrB inhibitors. The MIC of the quinazoline derivatives, Q203, LPZs and AX-35 was determined and expressed as the fold-change of MIC of each compound compared to M . tb H37Rv. (C) Bioenergetic analysis of M . tb upon Q203 and 11626252 treatment followed by the uncoupler CCCP to stimulate maximum respiration. The oxygen consumption rates of wild-type H37Rv, H37Rv Δ cydAB , and H37Rv Δ cyd KO with a QcrB (A317V) mutation (Q203 resistance SNP) was monitored as described in the Material and methods section. (D) Intracellular ATP levels in H37Rv and quinazoline resistant strains were measured in the absence and presence of BDQ, Q203, and 11626252 at 2.5× MIC after 24 h using BacTiterGlo (Promega). Data from two independent experiments are presented as mean ± SD. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests (*, P

    Article Snippet: M . tb H37Rv was obtained from BEI Resources (NR-123), M . tb H37Rv ΔcydAB [ ] and M . tb H37Rv ΔcydKO A317V [ ] were gifts from Dr. Digby Warner and Dr. Helena Boshoff, respectively.

    Techniques: Mutagenesis

    Mutation L356V in QcrA is responsible for quinazoline resistance in M . tb . Susceptibility to (A) 11626141, (B) 11626142, (C) 11626252 and (D) Rifampicin (RIF) was assessed in strains H37Rv-pYUB412 (black circle), H37Rv-Rv1777(Arg145Ser) (blue triangle), H37Rv-Rv2195(Leu356Val) (red square), and QuinR-M1 (green circle) by REMA. The graph represents the percentage of bacterial viability ± s.d. according to the drug concentrations. The experiment was performed in biological triplicates.

    Journal: PLoS Pathogens

    Article Title: New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis

    doi: 10.1371/journal.ppat.1008270

    Figure Lengend Snippet: Mutation L356V in QcrA is responsible for quinazoline resistance in M . tb . Susceptibility to (A) 11626141, (B) 11626142, (C) 11626252 and (D) Rifampicin (RIF) was assessed in strains H37Rv-pYUB412 (black circle), H37Rv-Rv1777(Arg145Ser) (blue triangle), H37Rv-Rv2195(Leu356Val) (red square), and QuinR-M1 (green circle) by REMA. The graph represents the percentage of bacterial viability ± s.d. according to the drug concentrations. The experiment was performed in biological triplicates.

    Article Snippet: M . tb H37Rv was obtained from BEI Resources (NR-123), M . tb H37Rv ΔcydAB [ ] and M . tb H37Rv ΔcydKO A317V [ ] were gifts from Dr. Digby Warner and Dr. Helena Boshoff, respectively.

    Techniques: Mutagenesis

    Transcriptome analysis of H37Rv exposed to 11626252. (A) Heat map representing top significantly differentially regulated M . tb genes ( P adj ≤ 0.05) after exposure of two independent cultures of M . tb H37Rv to 11626252 at 10X and 30X MIC for 4 h. The colour scale indicates differential regulation as log2 fold-change of H37Rv with 11626252 treatment relative to H37Rv with vehicle control, DMSO. Upregulation is indicated in red, downregulation is in green, and insignificant log2 fold change values for the condition are in black. Data are from two independent experiments. (B) Global transcriptome response and involvement of different metabolic responses, based on TubercuList classification ( https://mycobrowser.epfl.ch/ ). (C) Fold-change expression of cydB and lipU determined by qRT-PCR. Bars in black, light grey and white represent the gene expression compared to H37Rv-DMSO, QuinR1-M1 in the presence of DMSO, and QuinR-M2 with DMSO, respectively. Data from two independent cultures are presented as the mean relative expression ± s.d. Statistical analysis was performed using two-way analysis of variance ( ANOVA) with Tukey’s multiple-comparison tests (*, P

    Journal: PLoS Pathogens

    Article Title: New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis

    doi: 10.1371/journal.ppat.1008270

    Figure Lengend Snippet: Transcriptome analysis of H37Rv exposed to 11626252. (A) Heat map representing top significantly differentially regulated M . tb genes ( P adj ≤ 0.05) after exposure of two independent cultures of M . tb H37Rv to 11626252 at 10X and 30X MIC for 4 h. The colour scale indicates differential regulation as log2 fold-change of H37Rv with 11626252 treatment relative to H37Rv with vehicle control, DMSO. Upregulation is indicated in red, downregulation is in green, and insignificant log2 fold change values for the condition are in black. Data are from two independent experiments. (B) Global transcriptome response and involvement of different metabolic responses, based on TubercuList classification ( https://mycobrowser.epfl.ch/ ). (C) Fold-change expression of cydB and lipU determined by qRT-PCR. Bars in black, light grey and white represent the gene expression compared to H37Rv-DMSO, QuinR1-M1 in the presence of DMSO, and QuinR-M2 with DMSO, respectively. Data from two independent cultures are presented as the mean relative expression ± s.d. Statistical analysis was performed using two-way analysis of variance ( ANOVA) with Tukey’s multiple-comparison tests (*, P

    Article Snippet: M . tb H37Rv was obtained from BEI Resources (NR-123), M . tb H37Rv ΔcydAB [ ] and M . tb H37Rv ΔcydKO A317V [ ] were gifts from Dr. Digby Warner and Dr. Helena Boshoff, respectively.

    Techniques: Expressing, Quantitative RT-PCR

    11626252 is bacteriostatic but bactericidal in the absence of the bd oxidase. H37Rv, ΔcydAB and the complemented strain were exposed to increasing concentrations (1X MIC-32X MIC) of 11626252 for 7 days. Bacteria were plated on 7H10-OADC on day 0 (D0) to determine the initial number of CFU. After 7 days of exposure, untreated bacteria (D7) were plated for CFU enumeration. Results are expressed as the mean log 10 CFU/mL ± s.d.of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis

    doi: 10.1371/journal.ppat.1008270

    Figure Lengend Snippet: 11626252 is bacteriostatic but bactericidal in the absence of the bd oxidase. H37Rv, ΔcydAB and the complemented strain were exposed to increasing concentrations (1X MIC-32X MIC) of 11626252 for 7 days. Bacteria were plated on 7H10-OADC on day 0 (D0) to determine the initial number of CFU. After 7 days of exposure, untreated bacteria (D7) were plated for CFU enumeration. Results are expressed as the mean log 10 CFU/mL ± s.d.of two independent experiments.

    Article Snippet: M . tb H37Rv was obtained from BEI Resources (NR-123), M . tb H37Rv ΔcydAB [ ] and M . tb H37Rv ΔcydKO A317V [ ] were gifts from Dr. Digby Warner and Dr. Helena Boshoff, respectively.

    Techniques:

    phSGM2 accurately reported the STR MIC of a RIF r H37Rv variant. RIF r cells were added to the positive control and to STR- and RIF-containing wells of a Trek Sensititre dish and then infected with phSGM2. cSML generation was reduced to background levels

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

    doi: 10.1128/AAC.03135-14

    Figure Lengend Snippet: phSGM2 accurately reported the STR MIC of a RIF r H37Rv variant. RIF r cells were added to the positive control and to STR- and RIF-containing wells of a Trek Sensititre dish and then infected with phSGM2. cSML generation was reduced to background levels

    Article Snippet: The cSML was not detected in samples containing ∼105 CFU of either mc2 155 or H37Rv ( ) but was observed in phage alone controls.

    Techniques: Variant Assay, Positive Control, Infection

    phSGM2 detected the presence of 10% RIF r cells in an otherwise drug-susceptible population. H37Rv cells, RIF r cells, or mixtures of both containing 0.1, 1, or 10% RIF r cells were either left untreated or exposed to RIF, STR, or PNB and then infected with

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

    doi: 10.1128/AAC.03135-14

    Figure Lengend Snippet: phSGM2 detected the presence of 10% RIF r cells in an otherwise drug-susceptible population. H37Rv cells, RIF r cells, or mixtures of both containing 0.1, 1, or 10% RIF r cells were either left untreated or exposed to RIF, STR, or PNB and then infected with

    Article Snippet: The cSML was not detected in samples containing ∼105 CFU of either mc2 155 or H37Rv ( ) but was observed in phage alone controls.

    Techniques: Infection

    phSGM2 accurately detected susceptibility and resistance to all classes of anti-TB drugs. (A and B) cSML generation in H37Rv cells infected by phSGM2 and exposed to first-line (A) and second-line (B) anti-TB drugs. (C) phSGM2 detected resistance to RIF

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

    doi: 10.1128/AAC.03135-14

    Figure Lengend Snippet: phSGM2 accurately detected susceptibility and resistance to all classes of anti-TB drugs. (A and B) cSML generation in H37Rv cells infected by phSGM2 and exposed to first-line (A) and second-line (B) anti-TB drugs. (C) phSGM2 detected resistance to RIF

    Article Snippet: The cSML was not detected in samples containing ∼105 CFU of either mc2 155 or H37Rv ( ) but was observed in phage alone controls.

    Techniques: Infection

    Effect of infection in mice and rabbits with H37Rv and H37Rv::pPET1. (A) Growth of H37Rv and H37Rv::pPET1 in infected mouse lungs and spleen. Tissue homogenates from H37Rv::pPET1-infected mice were plated also on hygromycin-containing medium to determine

    Journal: Infection and Immunity

    Article Title: The Phenolic Glycolipid of Mycobacterium tuberculosis Differentially Modulates the Early Host Cytokine Response but Does Not in Itself Confer Hypervirulence

    doi: 10.1128/IAI.01663-07

    Figure Lengend Snippet: Effect of infection in mice and rabbits with H37Rv and H37Rv::pPET1. (A) Growth of H37Rv and H37Rv::pPET1 in infected mouse lungs and spleen. Tissue homogenates from H37Rv::pPET1-infected mice were plated also on hygromycin-containing medium to determine

    Article Snippet: These results suggested that PGL-tb production by H37Rv had only a modest effect on the production of specific cytokines and did not produce a generalized suppression of Th1 immunity.

    Techniques: Infection, Mouse Assay

    H37Rv and H37Rv::pPET1: in vitro growth in monocytes and cytokine production by infected PBMCs. (A) Growth in human monocytes in vitro of H37Rv and PGL-tb-producing H37Rv::pPET1. Mtb, M. tuberculosis . (B) Cytokine levels in supernatants from PBMCs infected

    Journal: Infection and Immunity

    Article Title: The Phenolic Glycolipid of Mycobacterium tuberculosis Differentially Modulates the Early Host Cytokine Response but Does Not in Itself Confer Hypervirulence

    doi: 10.1128/IAI.01663-07

    Figure Lengend Snippet: H37Rv and H37Rv::pPET1: in vitro growth in monocytes and cytokine production by infected PBMCs. (A) Growth in human monocytes in vitro of H37Rv and PGL-tb-producing H37Rv::pPET1. Mtb, M. tuberculosis . (B) Cytokine levels in supernatants from PBMCs infected

    Article Snippet: These results suggested that PGL-tb production by H37Rv had only a modest effect on the production of specific cytokines and did not produce a generalized suppression of Th1 immunity.

    Techniques: In Vitro, Infection

    Histology of infected BALB/c mouse lungs using hematoxylin and eosin stain. BALB/c mice were infected by the low-dose aerosol method with the H37Rv strain of M. tuberculosis (A and B), the Δ lsr2 strain (C and D), or the Δ lsr2 complemented strain (E and F). Low-magnification photomicrographs (A, C, and E) or higher-magnification photomicrographs (B, D, and E) are shown.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Histology of infected BALB/c mouse lungs using hematoxylin and eosin stain. BALB/c mice were infected by the low-dose aerosol method with the H37Rv strain of M. tuberculosis (A and B), the Δ lsr2 strain (C and D), or the Δ lsr2 complemented strain (E and F). Low-magnification photomicrographs (A, C, and E) or higher-magnification photomicrographs (B, D, and E) are shown.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Infection, H&E Stain, Mouse Assay

    Survival of the Δ lsr2 strain after addition of H 2 O 2 or mitomycin C during aerobic growth or in an anaerobic model. Cultures of H37Rv (white bars), the Δ lsr2 strain (horizontally striped bars), the Δ lsr2 complemented strain (diagonally striped bars), or the lsr2 overexpression strain (checkered bars) were exposed to water, 5 mM H 2 O 2 , or 50 nM mitomycin C (MitC) during aerobic growth (A), and the most probable number (MPN) was determined after 48 h. (B) Cultures were exposed to either water, 3 mM H 2 O 2 , or 50 nM mitomycin C at day 14 in a RAD model, and the MPN was determined after 5 days. Panels C and D are the calculated percentages of survival from panels A and B, respectively.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Survival of the Δ lsr2 strain after addition of H 2 O 2 or mitomycin C during aerobic growth or in an anaerobic model. Cultures of H37Rv (white bars), the Δ lsr2 strain (horizontally striped bars), the Δ lsr2 complemented strain (diagonally striped bars), or the lsr2 overexpression strain (checkered bars) were exposed to water, 5 mM H 2 O 2 , or 50 nM mitomycin C (MitC) during aerobic growth (A), and the most probable number (MPN) was determined after 48 h. (B) Cultures were exposed to either water, 3 mM H 2 O 2 , or 50 nM mitomycin C at day 14 in a RAD model, and the MPN was determined after 5 days. Panels C and D are the calculated percentages of survival from panels A and B, respectively.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Over Expression

    Growth and survival of the Δ lsr2 strain in an anaerobic model. Cultures of H37Rv (squares and white bars), the Δ lsr2 strain (open triangles and horizontally striped bars), the Δ lsr2 complemented strain (diamonds and diagonally striped bars), or the lsr2 overexpression strain (closed triangles and checkered bars) were cultured in the RAD anaerobic model. (A) OD 600 measurements were taken during the course of the RAD model. (B) OD 600 measurements during adaptation from aerobic to anaerobic phase of the model. (C) Viability MPN analysis at 0, 7, 14, 21, 28, 35, and 42 days in the RAD model.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Growth and survival of the Δ lsr2 strain in an anaerobic model. Cultures of H37Rv (squares and white bars), the Δ lsr2 strain (open triangles and horizontally striped bars), the Δ lsr2 complemented strain (diamonds and diagonally striped bars), or the lsr2 overexpression strain (closed triangles and checkered bars) were cultured in the RAD anaerobic model. (A) OD 600 measurements were taken during the course of the RAD model. (B) OD 600 measurements during adaptation from aerobic to anaerobic phase of the model. (C) Viability MPN analysis at 0, 7, 14, 21, 28, 35, and 42 days in the RAD model.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Over Expression, Cell Culture

    Survival of the Δ lsr2 strain after aerosol infection in a BALB/c mouse model. Six- to 8-week-old BALB/c mice were infected via aerosol infection with either H37Rv (squares), the Δ lsr2 strain (open triangles), or the Δ lsr2 complemented strain (diamonds). Lungs (A) or spleens (B) were harvested at 1, 14, and 28 days, and genome equivalents (GE) were determined using qPCR with primer/probe sets for both Rv1738 and Rv2626. Genome equivalents were averaged together for both primer/probe sets. (C) CFU counts were obtained from lung and spleen samples for the wild type (white bars) and the Δ lsr2 complemented strain (diagonally striped bars). (D) Disease scores were assessed for each mouse, and averages are shown for the wild type (Rv [white bars]), the Δ lsr2 strain (LKO), or the Δ lsr2 complemented strain (LCO [diagonally striped bars]). Five mice were used for each time point. An asterisk denotes differences that are statistically significant.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Survival of the Δ lsr2 strain after aerosol infection in a BALB/c mouse model. Six- to 8-week-old BALB/c mice were infected via aerosol infection with either H37Rv (squares), the Δ lsr2 strain (open triangles), or the Δ lsr2 complemented strain (diamonds). Lungs (A) or spleens (B) were harvested at 1, 14, and 28 days, and genome equivalents (GE) were determined using qPCR with primer/probe sets for both Rv1738 and Rv2626. Genome equivalents were averaged together for both primer/probe sets. (C) CFU counts were obtained from lung and spleen samples for the wild type (white bars) and the Δ lsr2 complemented strain (diagonally striped bars). (D) Disease scores were assessed for each mouse, and averages are shown for the wild type (Rv [white bars]), the Δ lsr2 strain (LKO), or the Δ lsr2 complemented strain (LCO [diagonally striped bars]). Five mice were used for each time point. An asterisk denotes differences that are statistically significant.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Infection, Mouse Assay, Real-time Polymerase Chain Reaction

    Recovery from anaerobiosis. H37Rv (squares), the Δ lsr2 strain (open triangles), the Δ lsr2 complemented strain (diamonds), or the lsr2 overexpression strain (closed triangles) was added to RAD model tubes. After 7 days (A) or 14 days (B), tubes were opened as indicated by the arrows, all but 5 ml of culture was removed to ensure proper aeration, and the OD 600 was obtained every 24 h.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Recovery from anaerobiosis. H37Rv (squares), the Δ lsr2 strain (open triangles), the Δ lsr2 complemented strain (diamonds), or the lsr2 overexpression strain (closed triangles) was added to RAD model tubes. After 7 days (A) or 14 days (B), tubes were opened as indicated by the arrows, all but 5 ml of culture was removed to ensure proper aeration, and the OD 600 was obtained every 24 h.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Over Expression

    Oxygen consumption during adaptation to anaerobiosis as measured by methylene blue decolorization. H37Rv (squares), the Δ lsr2 strain (open triangles), the Δ lsr2 complemented strain (diamonds), and the lsr2 overexpression strain (closed triangles) were added to a RAD model. Methylene blue (MB) was added (final concentration, 9 µg/ml) at time zero, and the OD 600 measurement was taken at regular intervals until methylene blue decolorization was complete.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Oxygen consumption during adaptation to anaerobiosis as measured by methylene blue decolorization. H37Rv (squares), the Δ lsr2 strain (open triangles), the Δ lsr2 complemented strain (diamonds), and the lsr2 overexpression strain (closed triangles) were added to a RAD model. Methylene blue (MB) was added (final concentration, 9 µg/ml) at time zero, and the OD 600 measurement was taken at regular intervals until methylene blue decolorization was complete.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Over Expression, Concentration Assay

    Growth of the Δ lsr2 strain under hypoxic, normoxic, and hyperoxic oxygen tension. Cultures of H37Rv (squares and white bars), the Δ lsr2 strain (open triangles and horizontally striped bars), the Δ lsr2 complemented strain (diamonds and diagonally striped bars), or the lsr2 overexpression strain (closed triangles and checkered bars) were grown in DTA medium in either a chamber containing 2% oxygen (A), under atmospheric oxygen (~18% oxygen) (B), or a chamber containing 60% oxygen (C). (D) The doubling time was calculated from between day 1 and day 3 for each strain.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    doi: 10.1128/mBio.01106-14

    Figure Lengend Snippet: Growth of the Δ lsr2 strain under hypoxic, normoxic, and hyperoxic oxygen tension. Cultures of H37Rv (squares and white bars), the Δ lsr2 strain (open triangles and horizontally striped bars), the Δ lsr2 complemented strain (diamonds and diagonally striped bars), or the lsr2 overexpression strain (closed triangles and checkered bars) were grown in DTA medium in either a chamber containing 2% oxygen (A), under atmospheric oxygen (~18% oxygen) (B), or a chamber containing 60% oxygen (C). (D) The doubling time was calculated from between day 1 and day 3 for each strain.

    Article Snippet: The mice were then aerosol infected with around 4,000 bacilli (per mouse) of H37Rv, the Δlsr2 strain, or the Δlsr2 complemented strain by the Glas-Col inhalation exposure system as previously described ( ).

    Techniques: Over Expression

    Most recent common ancestors and deletion events during the evolution of the Mj sublineage of Nunavik. Because similar estimates with overlapping 95% highest posterior density intervals were obtained in all three MRCA analyses, MRCA dates obtained via analysis 1 are presented for simplicity. Arrows indicate the positions and annotations of the H37Rv reference genome. A phylogenetic cluster was defined as at least two genomes sharing a minimum of two single-nucleotide polymorphic loci. Gray and colored circles represent the common ancestors and phylogenetic clusters based on identified SNPs, respectively. Isolate 58385 had a unique single-nucleotide polymorphism profile (i.e., was not clustered) and, because the posterior density for the divergence date of this isolate was

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

    Article Title: Population genomics of Mycobacterium tuberculosis in the Inuit

    doi: 10.1073/pnas.1507071112

    Figure Lengend Snippet: Most recent common ancestors and deletion events during the evolution of the Mj sublineage of Nunavik. Because similar estimates with overlapping 95% highest posterior density intervals were obtained in all three MRCA analyses, MRCA dates obtained via analysis 1 are presented for simplicity. Arrows indicate the positions and annotations of the H37Rv reference genome. A phylogenetic cluster was defined as at least two genomes sharing a minimum of two single-nucleotide polymorphic loci. Gray and colored circles represent the common ancestors and phylogenetic clusters based on identified SNPs, respectively. Isolate 58385 had a unique single-nucleotide polymorphism profile (i.e., was not clustered) and, because the posterior density for the divergence date of this isolate was

    Article Snippet: Reads were aligned to H37Rv [National Center for Biotechnology Information (NCBI) accession no. ] and compared as previously described ( ).

    Techniques:

    Maximum likelihood tree of 163 M. tuberculosis isolates from Nunavik. Phylogenetic clusters were identified based on 1,288 single-nucleotide polymorphic loci compared with H37Rv. Solid and dashed lines indicate isolates of the Mj and Mn sublineages, respectively. Colored shapes represent the reference genome (bordered black square) and the villages of Nunavik: A (bordered blue triangle), B (full orange square), C (bordered purple circle), D (full green diamond), E (bordered purple diamond), K (full pink triangle), and other (full green circle). *Genome with a unique single-nucleotide polymorphism profile. # . Years of diagnosis are indicated. Bootstrap support from 1,000 replicates is shown. Branches supported by less than 80% of bootstrap replicates are collapsed.

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

    Article Title: Population genomics of Mycobacterium tuberculosis in the Inuit

    doi: 10.1073/pnas.1507071112

    Figure Lengend Snippet: Maximum likelihood tree of 163 M. tuberculosis isolates from Nunavik. Phylogenetic clusters were identified based on 1,288 single-nucleotide polymorphic loci compared with H37Rv. Solid and dashed lines indicate isolates of the Mj and Mn sublineages, respectively. Colored shapes represent the reference genome (bordered black square) and the villages of Nunavik: A (bordered blue triangle), B (full orange square), C (bordered purple circle), D (full green diamond), E (bordered purple diamond), K (full pink triangle), and other (full green circle). *Genome with a unique single-nucleotide polymorphism profile. # . Years of diagnosis are indicated. Bootstrap support from 1,000 replicates is shown. Branches supported by less than 80% of bootstrap replicates are collapsed.

    Article Snippet: Reads were aligned to H37Rv [National Center for Biotechnology Information (NCBI) accession no. ] and compared as previously described ( ).

    Techniques:

    Maximum likelihood tree of 163 M. tuberculosis isolates from Nunavik and 21 representative genomes of lineages 1–7. Phylogenetic clusters based on 9,016 single-nucleotide polymorphic loci identified across 184 genomes compared with H37Rv (solid black circle). The scale bars represent the number of substitutions per site. Bootstrap values from 1,000 replicates are shown for branches within the Mj and Mn sublineages. For clarity, only values ≥98 are shown.

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

    Article Title: Population genomics of Mycobacterium tuberculosis in the Inuit

    doi: 10.1073/pnas.1507071112

    Figure Lengend Snippet: Maximum likelihood tree of 163 M. tuberculosis isolates from Nunavik and 21 representative genomes of lineages 1–7. Phylogenetic clusters based on 9,016 single-nucleotide polymorphic loci identified across 184 genomes compared with H37Rv (solid black circle). The scale bars represent the number of substitutions per site. Bootstrap values from 1,000 replicates are shown for branches within the Mj and Mn sublineages. For clarity, only values ≥98 are shown.

    Article Snippet: Reads were aligned to H37Rv [National Center for Biotechnology Information (NCBI) accession no. ] and compared as previously described ( ).

    Techniques:

    Live M. tuberculosis infection causes dendritic cell death and DNA fragmentation, without nuclear fragmentation . A - B . Dendritic cells (DCs) were infected, at MOI 10 with live/dead H37Ra or live/dead H37Rv. (U = uninfected, LH37Ra = live H37Ra, sH37Ra = streptomycin-killed H37Ra, LH37Rv = live H37Rv, iH37Rv = γ-irradiated H37Rv.) Cell death was measured by propidium iodide exclusion (A) 72 h post-infection or (B) 24 h post-infection on a GE IN Cell Analyzer 1000. (A - B) are means (± SEM) of 3 pooled donors. * p

    Journal: BMC Microbiology

    Article Title: Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    doi: 10.1186/1471-2180-11-237

    Figure Lengend Snippet: Live M. tuberculosis infection causes dendritic cell death and DNA fragmentation, without nuclear fragmentation . A - B . Dendritic cells (DCs) were infected, at MOI 10 with live/dead H37Ra or live/dead H37Rv. (U = uninfected, LH37Ra = live H37Ra, sH37Ra = streptomycin-killed H37Ra, LH37Rv = live H37Rv, iH37Rv = γ-irradiated H37Rv.) Cell death was measured by propidium iodide exclusion (A) 72 h post-infection or (B) 24 h post-infection on a GE IN Cell Analyzer 1000. (A - B) are means (± SEM) of 3 pooled donors. * p

    Article Snippet: The cells were fixed for 10 min (H37Ra) or 24 h (H37Rv) in 2% paraformaldehyde (Sigma), applied to glass slides and left to air dry overnight.

    Techniques: Infection, Irradiation

    Dendritic cells mature after they phagocytose M. tuberculosis . A . Human monocytes were separated from buffy coats by plastic adherence and cultured for 6 days in the presence of recombinant human IL-4 (40 ng/ml) and GM-CSF (50 ng/ml) to allow differentiation to DCs. Cells were analysed for CD14 and DC-SIGN expression by flow cytometry. DCs were CD14 - and DC-SIGN + (typically > 85% of gated cells; both before and after infection with Mtb). Plots show uninfected, immature DCs after 6 days of cytokine treatment from 1 representative donor of 3.. B . DCs were infected with live H37Ra at MOI 1 for 24 h and visualised by light microscopy. C . DCs were infected with live Mtb H37Rv at MOI 10 overnight. Bacteria were stained with auramine and nuclei with Hoechst and were visualised by confocal microscopy. Similar results were obtained with iH37Rv, live H37Ra and streptomycin-killed H37Ra (data not shown). D . DCs were infected with live Mtb H37Ra or streptomycin-killed H37Ra at MOI 1 for 48 h. Surface expression of CD83 and CD86 was assessed by flow cytometry. The histograms show 1 representative donor of 3.

    Journal: BMC Microbiology

    Article Title: Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    doi: 10.1186/1471-2180-11-237

    Figure Lengend Snippet: Dendritic cells mature after they phagocytose M. tuberculosis . A . Human monocytes were separated from buffy coats by plastic adherence and cultured for 6 days in the presence of recombinant human IL-4 (40 ng/ml) and GM-CSF (50 ng/ml) to allow differentiation to DCs. Cells were analysed for CD14 and DC-SIGN expression by flow cytometry. DCs were CD14 - and DC-SIGN + (typically > 85% of gated cells; both before and after infection with Mtb). Plots show uninfected, immature DCs after 6 days of cytokine treatment from 1 representative donor of 3.. B . DCs were infected with live H37Ra at MOI 1 for 24 h and visualised by light microscopy. C . DCs were infected with live Mtb H37Rv at MOI 10 overnight. Bacteria were stained with auramine and nuclei with Hoechst and were visualised by confocal microscopy. Similar results were obtained with iH37Rv, live H37Ra and streptomycin-killed H37Ra (data not shown). D . DCs were infected with live Mtb H37Ra or streptomycin-killed H37Ra at MOI 1 for 48 h. Surface expression of CD83 and CD86 was assessed by flow cytometry. The histograms show 1 representative donor of 3.

    Article Snippet: The cells were fixed for 10 min (H37Ra) or 24 h (H37Rv) in 2% paraformaldehyde (Sigma), applied to glass slides and left to air dry overnight.

    Techniques: Cell Culture, Recombinant, Expressing, Flow Cytometry, Cytometry, Infection, Light Microscopy, Staining, Confocal Microscopy

    Humoral immune response in BAL and plasma after BCG immunization. Mtb-responsive antibody responses were assessed in BAL and plasma after BCG immunization. Mtb WCL-specific IgG, IgA and IgM antibody titres were measured from individual NHPs at various time points before and after BCG immunization. Shown are end-point titres for IgG and IgA and mid-point titres for IgM (in which the end point was not reached) a , Antibody titres in tenfold-concentrated BAL fluid (cohorts 1–4, n = 11–13 macaques except at weeks 2, 20 and 24, cohort 4 only, n = 3 macaques). In concentrated BAL fluid, antigen-responsive IgG, IgA and IgM were detected only in IV-BCG-immunized NHPs and returned to pre-vaccination levels by the time of challenge. b , Antibody titres in plasma ( n = 11–13 macaques). In plasma, both ID high and IV BCG elicited increased IgG and IgA antibody responses compared to ID low BCG. Data are geometric mean and s.d.; dashed line indicates assay limit of detection. A Kruskal–Wallis test was used to compare all vaccine groups to ID low at weeks 4, 16 and 24 (BAL) or weeks 4 and 24 (plasma); P values are from Dunn’s multiple comparison test (colour-coded to vaccine). Source Data

    Journal: Nature

    Article Title: Prevention of tuberculosis in macaques after intravenous BCG immunization

    doi: 10.1038/s41586-019-1817-8

    Figure Lengend Snippet: Humoral immune response in BAL and plasma after BCG immunization. Mtb-responsive antibody responses were assessed in BAL and plasma after BCG immunization. Mtb WCL-specific IgG, IgA and IgM antibody titres were measured from individual NHPs at various time points before and after BCG immunization. Shown are end-point titres for IgG and IgA and mid-point titres for IgM (in which the end point was not reached) a , Antibody titres in tenfold-concentrated BAL fluid (cohorts 1–4, n = 11–13 macaques except at weeks 2, 20 and 24, cohort 4 only, n = 3 macaques). In concentrated BAL fluid, antigen-responsive IgG, IgA and IgM were detected only in IV-BCG-immunized NHPs and returned to pre-vaccination levels by the time of challenge. b , Antibody titres in plasma ( n = 11–13 macaques). In plasma, both ID high and IV BCG elicited increased IgG and IgA antibody responses compared to ID low BCG. Data are geometric mean and s.d.; dashed line indicates assay limit of detection. A Kruskal–Wallis test was used to compare all vaccine groups to ID low at weeks 4, 16 and 24 (BAL) or weeks 4 and 24 (plasma); P values are from Dunn’s multiple comparison test (colour-coded to vaccine). Source Data

    Article Snippet: For T cell stimulation assays, 1–5 million viable cells were plated in 96-well V-bottom plates (Corning) in R10 and incubated with R10 alone (background), or with 20 μg ml−1 tuberculin PPD (Statens Serum Institut, Copenhagen, Denmark), 20 μg ml−1 H37Rv Mtb WCL (BEI Resources), or 1 μg ml−1 each of ESAT-6 and CFP-10 peptide pools (provided by Aeras, Rockville, MD) for 2 h before adding 10 μg ml−1 BD GolgiPlug (BD Biosciences).

    Techniques:

    Determination of immune responses and BCG in tissues 1 month after immunization. NHPs (cohort 6, n = 2) were immunized with 5 × 10 7 BCG CFUs (ID high ( a ), IV ( b ), AE ( c ), or EB ( d )); BCG CFUs and antigen-responsive T cells were measured in various tissues 1 month later. Before euthanasia, a fluorochrome-conjugated anti-CD45 antibody was injected intravenously (ivCD45) such that circulating (intravascular) leukocytes were uniformly stained (ivCD45 + ) while leukocytes in the tissue remained protected from staining (ivCD45 − ). To investigate whether antigen-responsive (IFNγ + ) CD4 T cells were located in the ivCD45 − lung tissue compartment, cells isolated from lung lobes were re-stimulated in vitro with Mtb WCL and analysed by intracellular cytokine staining. FACS plots show memory CD4 T cells in all tissues collected from one of two NHPs per BCG regimen, organized by type/location (systemic, peripheral LN, lung LN, BAL and lung lobes). The BAL and lung responses from the IV BCG NHPs, shown in the bottom row of b , is reproduced from Fig. 4b . Pre-infusion PBMC indicates PBMCs isolated from whole blood collected just before anti-CD45 injection. Bar graphs show the number of BCG CFUs in each respective tissue for each animal (colour-coded by vaccine), if detected. Source Data

    Journal: Nature

    Article Title: Prevention of tuberculosis in macaques after intravenous BCG immunization

    doi: 10.1038/s41586-019-1817-8

    Figure Lengend Snippet: Determination of immune responses and BCG in tissues 1 month after immunization. NHPs (cohort 6, n = 2) were immunized with 5 × 10 7 BCG CFUs (ID high ( a ), IV ( b ), AE ( c ), or EB ( d )); BCG CFUs and antigen-responsive T cells were measured in various tissues 1 month later. Before euthanasia, a fluorochrome-conjugated anti-CD45 antibody was injected intravenously (ivCD45) such that circulating (intravascular) leukocytes were uniformly stained (ivCD45 + ) while leukocytes in the tissue remained protected from staining (ivCD45 − ). To investigate whether antigen-responsive (IFNγ + ) CD4 T cells were located in the ivCD45 − lung tissue compartment, cells isolated from lung lobes were re-stimulated in vitro with Mtb WCL and analysed by intracellular cytokine staining. FACS plots show memory CD4 T cells in all tissues collected from one of two NHPs per BCG regimen, organized by type/location (systemic, peripheral LN, lung LN, BAL and lung lobes). The BAL and lung responses from the IV BCG NHPs, shown in the bottom row of b , is reproduced from Fig. 4b . Pre-infusion PBMC indicates PBMCs isolated from whole blood collected just before anti-CD45 injection. Bar graphs show the number of BCG CFUs in each respective tissue for each animal (colour-coded by vaccine), if detected. Source Data

    Article Snippet: For T cell stimulation assays, 1–5 million viable cells were plated in 96-well V-bottom plates (Corning) in R10 and incubated with R10 alone (background), or with 20 μg ml−1 tuberculin PPD (Statens Serum Institut, Copenhagen, Denmark), 20 μg ml−1 H37Rv Mtb WCL (BEI Resources), or 1 μg ml−1 each of ESAT-6 and CFP-10 peptide pools (provided by Aeras, Rockville, MD) for 2 h before adding 10 μg ml−1 BD GolgiPlug (BD Biosciences).

    Techniques: Injection, Staining, Isolation, In Vitro, FACS

    Immune response to BCG 6 months after vaccination. Analysis of tissue T cell responses, lung cell counts, immunohistochemistry, splenic volume and PET–CT scans was performed 6 months after BCG vaccination. a – c , A separate cohort (cohort 3, n = 3 macaques) was vaccinated with BCG in parallel to the challenge study with the purpose of assessing immune responses in various tissues 6 months after BCG (the time point at which macaques would be challenged). a , b , Frequency of memory CD4 ( a ) and CD8 ( b ) T cells producing any combination of IFNγ, IL-2, TNF, or IL-17 in response to Mtb WCL stimulation in the PBMC, spleen, bone marrow, peripheral LN, lung LN, lung tissue and BAL. Six months after IV BCG, immunized NHPs maintained increased frequencies of antigen-responsive T cells in spleen, BAL and lung lobes. Individual LN and lung lobe responses were averaged per macaque. Data points are individual macaques with symbols matched across tissues within a vaccine group; horizontal bar indicates the mean response. c , Number of cells recovered per gram of lung tissue for each NHP; the increased numbers of total cells observed at 1 month post-BCG (Fig. 3d ) were not detected at 6 months post-BCG. Data are shown as the median of 3 macaques per group (solid symbols, counts from six lung lobes per animal are averaged) or as counts for individual lung lobes for each animal (open symbols; lobes from the same animal have matched symbols). Kruskal–Wallis test was used, and P values represent Dunn’s multiple comparison test comparing each vaccine group to the ID low group. d , Quantification of CD3 + , CD20 + , CD11c + cells from two lung sections (matched symbols) from 1–2 macaques per group using Cell Profiler. e , Representative 1-mm 2 lung sections from 1–2 macaques per vaccine group were stained with H E or with antibodies against CD3 + T cells (red), CD20 + B cells (green), and CD11c + macrophages or dendritic cells (blue). Neither the increase in numbers of T cells and CD11c + cells nor the histopathological changes in lung sections from IV-BCG-immunized macaques observed at 1 month (Fig. 3e, f ) were detected 6 months after BCG vaccination. f , Spleen volume was calculated from CT scans of 44 NHPs (cohorts 1–3) just before Mtb challenge (6 months after BCG vaccination) and was not significantly different among vaccine routes (Kruskal–Wallis test, P = 0.1643). Dots represent individual animals. g , Axial (top) and coronal (bottom) PET–CT scans of two representative macaques ( n = 8–10) from each vaccine group 6 months after BCG, before Mtb infection. Animal ID numbers are shown below each set of scans. No detectable lung inflammation (FDG uptake) was observed in macaques from any vaccine group. Source Data

    Journal: Nature

    Article Title: Prevention of tuberculosis in macaques after intravenous BCG immunization

    doi: 10.1038/s41586-019-1817-8

    Figure Lengend Snippet: Immune response to BCG 6 months after vaccination. Analysis of tissue T cell responses, lung cell counts, immunohistochemistry, splenic volume and PET–CT scans was performed 6 months after BCG vaccination. a – c , A separate cohort (cohort 3, n = 3 macaques) was vaccinated with BCG in parallel to the challenge study with the purpose of assessing immune responses in various tissues 6 months after BCG (the time point at which macaques would be challenged). a , b , Frequency of memory CD4 ( a ) and CD8 ( b ) T cells producing any combination of IFNγ, IL-2, TNF, or IL-17 in response to Mtb WCL stimulation in the PBMC, spleen, bone marrow, peripheral LN, lung LN, lung tissue and BAL. Six months after IV BCG, immunized NHPs maintained increased frequencies of antigen-responsive T cells in spleen, BAL and lung lobes. Individual LN and lung lobe responses were averaged per macaque. Data points are individual macaques with symbols matched across tissues within a vaccine group; horizontal bar indicates the mean response. c , Number of cells recovered per gram of lung tissue for each NHP; the increased numbers of total cells observed at 1 month post-BCG (Fig. 3d ) were not detected at 6 months post-BCG. Data are shown as the median of 3 macaques per group (solid symbols, counts from six lung lobes per animal are averaged) or as counts for individual lung lobes for each animal (open symbols; lobes from the same animal have matched symbols). Kruskal–Wallis test was used, and P values represent Dunn’s multiple comparison test comparing each vaccine group to the ID low group. d , Quantification of CD3 + , CD20 + , CD11c + cells from two lung sections (matched symbols) from 1–2 macaques per group using Cell Profiler. e , Representative 1-mm 2 lung sections from 1–2 macaques per vaccine group were stained with H E or with antibodies against CD3 + T cells (red), CD20 + B cells (green), and CD11c + macrophages or dendritic cells (blue). Neither the increase in numbers of T cells and CD11c + cells nor the histopathological changes in lung sections from IV-BCG-immunized macaques observed at 1 month (Fig. 3e, f ) were detected 6 months after BCG vaccination. f , Spleen volume was calculated from CT scans of 44 NHPs (cohorts 1–3) just before Mtb challenge (6 months after BCG vaccination) and was not significantly different among vaccine routes (Kruskal–Wallis test, P = 0.1643). Dots represent individual animals. g , Axial (top) and coronal (bottom) PET–CT scans of two representative macaques ( n = 8–10) from each vaccine group 6 months after BCG, before Mtb infection. Animal ID numbers are shown below each set of scans. No detectable lung inflammation (FDG uptake) was observed in macaques from any vaccine group. Source Data

    Article Snippet: For T cell stimulation assays, 1–5 million viable cells were plated in 96-well V-bottom plates (Corning) in R10 and incubated with R10 alone (background), or with 20 μg ml−1 tuberculin PPD (Statens Serum Institut, Copenhagen, Denmark), 20 μg ml−1 H37Rv Mtb WCL (BEI Resources), or 1 μg ml−1 each of ESAT-6 and CFP-10 peptide pools (provided by Aeras, Rockville, MD) for 2 h before adding 10 μg ml−1 BD GolgiPlug (BD Biosciences).

    Techniques: Immunohistochemistry, Positron Emission Tomography, Staining, Infection

    Detection of T cells in lung tissue after IV BCG immunization. a , One month after BCG vaccination, tissue-derived versus blood-derived cells in lung were delineated by injecting NHPs with a fluorochrome-conjugated anti-CD45 antibody (ivCD45) to label leukocytes in the vasculature. NHPs (cohort 6, n = 2 macaques) received 5 × 10 7 CFUs BCG ID, IV, AE or endobronchially (EB) into the left lung. At necropsy, BCG CFUs were quantified in tissues and cells were stained immediately ex vivo for surface marker expression ( a ) or stimulated with Mtb whole-cell lysate (WCL) and stained for cytokine production ( b , c ). Plots show CD4 T cells from the BAL and lung lobes (RU, right upper; RM, right middle; RL, right lower; LU, left upper; LL, left lower) from one of two macaques per BCG regimen. a , Percentage of ivCD45 − (unstimulated) CD4 T cells expressing the tissue-resident/activation marker CD69; BCG CFUs (if detected) are indicated by red bars and right scale. b , Percentage of WCL-responsive (IFNγ + ) CD4 T cells in BAL and lung tissue (ivCD45 − ) and ( c ) the percentage of IFNγ + CD4 memory T cells expressing CD69 and CD103 after IV BCG vaccination. Source Data

    Journal: Nature

    Article Title: Prevention of tuberculosis in macaques after intravenous BCG immunization

    doi: 10.1038/s41586-019-1817-8

    Figure Lengend Snippet: Detection of T cells in lung tissue after IV BCG immunization. a , One month after BCG vaccination, tissue-derived versus blood-derived cells in lung were delineated by injecting NHPs with a fluorochrome-conjugated anti-CD45 antibody (ivCD45) to label leukocytes in the vasculature. NHPs (cohort 6, n = 2 macaques) received 5 × 10 7 CFUs BCG ID, IV, AE or endobronchially (EB) into the left lung. At necropsy, BCG CFUs were quantified in tissues and cells were stained immediately ex vivo for surface marker expression ( a ) or stimulated with Mtb whole-cell lysate (WCL) and stained for cytokine production ( b , c ). Plots show CD4 T cells from the BAL and lung lobes (RU, right upper; RM, right middle; RL, right lower; LU, left upper; LL, left lower) from one of two macaques per BCG regimen. a , Percentage of ivCD45 − (unstimulated) CD4 T cells expressing the tissue-resident/activation marker CD69; BCG CFUs (if detected) are indicated by red bars and right scale. b , Percentage of WCL-responsive (IFNγ + ) CD4 T cells in BAL and lung tissue (ivCD45 − ) and ( c ) the percentage of IFNγ + CD4 memory T cells expressing CD69 and CD103 after IV BCG vaccination. Source Data

    Article Snippet: For T cell stimulation assays, 1–5 million viable cells were plated in 96-well V-bottom plates (Corning) in R10 and incubated with R10 alone (background), or with 20 μg ml−1 tuberculin PPD (Statens Serum Institut, Copenhagen, Denmark), 20 μg ml−1 H37Rv Mtb WCL (BEI Resources), or 1 μg ml−1 each of ESAT-6 and CFP-10 peptide pools (provided by Aeras, Rockville, MD) for 2 h before adding 10 μg ml−1 BD GolgiPlug (BD Biosciences).

    Techniques: Derivative Assay, Staining, Ex Vivo, Marker, Expressing, Activation Assay

    Hematopoietic miR-33 deficiency enhances autophagy gene expression in the lung and reduces Mtb burden in mice Analysis of ( a ) gene expression and ( b ) bacterial counts in the lungs of wild type (WT→WT) and Mir33 −/− ( Mir33 −/− →WT) bone marrow chimeric mice infected with H37Rv Mtb for the indicated time post-infection (p.i). * P ≤0.05, ** P ≤0.005 (Student’s t-test ( a , b )). Data are from 5 mice ( a, b ; mean ± s.e.m).

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: Hematopoietic miR-33 deficiency enhances autophagy gene expression in the lung and reduces Mtb burden in mice Analysis of ( a ) gene expression and ( b ) bacterial counts in the lungs of wild type (WT→WT) and Mir33 −/− ( Mir33 −/− →WT) bone marrow chimeric mice infected with H37Rv Mtb for the indicated time post-infection (p.i). * P ≤0.05, ** P ≤0.005 (Student’s t-test ( a , b )). Data are from 5 mice ( a, b ; mean ± s.e.m).

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Expressing, Mouse Assay, Infection

    Inhibition of miR-33 and miR-33* enhances targeting of M. tuberculosis for autophagy and bacterial killing ( a ) Immunofluorescence imaging of peritoneal macrophages treated with control anti-miR, anti-miR-33, anti-miR-33*, or IFN-γ (200 units/mL) and infected with an Mtb H37Rv strain co-expressing mCherry and anhydrotetracycline-inducible GFP for 48 h. Metabolically active Mtb are GFP and mCherry positive, whereas inactive or non-viable bacteria are mCherry positive only. Scale bar = 25 μm. Quantification of bacterial viability in peritoneal macrophages infected with Mtb H37Rv or ΔesxA mutant strain shown on the right. ( b–c ) Quantification of Mtb survival in wild type (WT) or miR-33 −/− BMDMs treated with or without IFN-γ (200 units/mL) using ( b ) the dual fluorescence viability assay as in ( a ), or ( c ) colony forming units (CFU) 5 days post-infection. ( d ) Mtb survival in wild type (WT) or Atg16l1 −/− BMDMs treated with control anti-miR, anti-miR-33 or anti-miR-33*, measured by CFU 5 days post-infection. ( e ) Mtb survival in wild type (WT) or cGAS −/− BMDMs treated with control anti-miR, anti-miR-33 or anti-miR-33*, measured by CFU 5 days post-infection. NS, not significant; * P ≤0.05, **P≤0.005 (Two-way ANOVA ( a – e )). Data are from one experiment and are representative of 2 independent experiments (a-e; mean ± s.e.m) with similar findings.

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: Inhibition of miR-33 and miR-33* enhances targeting of M. tuberculosis for autophagy and bacterial killing ( a ) Immunofluorescence imaging of peritoneal macrophages treated with control anti-miR, anti-miR-33, anti-miR-33*, or IFN-γ (200 units/mL) and infected with an Mtb H37Rv strain co-expressing mCherry and anhydrotetracycline-inducible GFP for 48 h. Metabolically active Mtb are GFP and mCherry positive, whereas inactive or non-viable bacteria are mCherry positive only. Scale bar = 25 μm. Quantification of bacterial viability in peritoneal macrophages infected with Mtb H37Rv or ΔesxA mutant strain shown on the right. ( b–c ) Quantification of Mtb survival in wild type (WT) or miR-33 −/− BMDMs treated with or without IFN-γ (200 units/mL) using ( b ) the dual fluorescence viability assay as in ( a ), or ( c ) colony forming units (CFU) 5 days post-infection. ( d ) Mtb survival in wild type (WT) or Atg16l1 −/− BMDMs treated with control anti-miR, anti-miR-33 or anti-miR-33*, measured by CFU 5 days post-infection. ( e ) Mtb survival in wild type (WT) or cGAS −/− BMDMs treated with control anti-miR, anti-miR-33 or anti-miR-33*, measured by CFU 5 days post-infection. NS, not significant; * P ≤0.05, **P≤0.005 (Two-way ANOVA ( a – e )). Data are from one experiment and are representative of 2 independent experiments (a-e; mean ± s.e.m) with similar findings.

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Inhibition, Immunofluorescence, Imaging, Infection, Expressing, Metabolic Labelling, Mutagenesis, Fluorescence, Viability Assay

    miR-33 and miR-33* reduce fatty acid oxidation and promote lipid body formation in M. tuberculosis -infected macrophages (a) Oxygen consumption rate (OCR) of BMDMs cultured with γ-Mtb for 6h and sequentially treated with Oligo, FCCP and rotenone plus antimycin (Rtn/AA) at the indicated times (arrows), measured in real time and presented as change in pMoles per unit time. (b) Maximal OCR of WT or miR-33 −/− BMDMs cultured with γ-Mtb for 6h measured in real time and presented as change in pMoles per unit time (OCR). (c) Immunofluorescence imaging of BODIPY-stained lipid droplets in control anti-miR, anti-miR-33 or anti-miR-33* treated THP-1 macrophages infected with H37Rv for 24h. Scale bar = 25μm. ( d ) Immunofluorescence (IF) imaging of WT or miR-33 −/− macrophages incubated with BODIPY-FL-C 16 and BSA-conjugated oleic acid for 48 h to label nascent lipid droplets and then infected with DsRed-H37Rv Mtb for 6h. Scale bar = 25μm. ( e ) IF imaging of BODIPY-stained lipid droplets in ctrl anti-miR, anti-miR33 or anti-miR33* treated macrophages, incubated with or without an inhibitor of lysosomal acid lipase (LALi, 10μM) for 8h. Scale bar = 10μm. NS, not significant, * P ≤0.05, **P≤0.005 (Student’s t-test ( b ), one-way ANOVA ( e )). Data are from one experiment representative of 3 ( a , b ; mean ± s.e.m, and d ) or 2 ( c and e; mean ± s.e.m) independent experiments with similar results.

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: miR-33 and miR-33* reduce fatty acid oxidation and promote lipid body formation in M. tuberculosis -infected macrophages (a) Oxygen consumption rate (OCR) of BMDMs cultured with γ-Mtb for 6h and sequentially treated with Oligo, FCCP and rotenone plus antimycin (Rtn/AA) at the indicated times (arrows), measured in real time and presented as change in pMoles per unit time. (b) Maximal OCR of WT or miR-33 −/− BMDMs cultured with γ-Mtb for 6h measured in real time and presented as change in pMoles per unit time (OCR). (c) Immunofluorescence imaging of BODIPY-stained lipid droplets in control anti-miR, anti-miR-33 or anti-miR-33* treated THP-1 macrophages infected with H37Rv for 24h. Scale bar = 25μm. ( d ) Immunofluorescence (IF) imaging of WT or miR-33 −/− macrophages incubated with BODIPY-FL-C 16 and BSA-conjugated oleic acid for 48 h to label nascent lipid droplets and then infected with DsRed-H37Rv Mtb for 6h. Scale bar = 25μm. ( e ) IF imaging of BODIPY-stained lipid droplets in ctrl anti-miR, anti-miR33 or anti-miR33* treated macrophages, incubated with or without an inhibitor of lysosomal acid lipase (LALi, 10μM) for 8h. Scale bar = 10μm. NS, not significant, * P ≤0.05, **P≤0.005 (Student’s t-test ( b ), one-way ANOVA ( e )). Data are from one experiment representative of 3 ( a , b ; mean ± s.e.m, and d ) or 2 ( c and e; mean ± s.e.m) independent experiments with similar results.

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Infection, Cell Culture, Immunofluorescence, Imaging, Staining, Incubation

    Regulation of human and mouse autophagy gene targets by miR-33 and miR-33* ( a–b ) 3’UTR-luciferase reporter activity of human autophagy gene targets in HEK293 cells transfected with control (ctrl) mimics or (a) hsa-miR-33a or (b) hsa-miR-33a* (left panels). Western blotting of lysates from human THP-1 macrophages treated with control (ctrl) mimics or (a) hsa-miR-33a or (b) hsa-miR-33a* (right panels)., HSP90 is shown as an internal control (right). ( c–d ) 3’UTR-luciferase reporter activity of mouse autophagy gene targets in HEK293 cells transfected with control mimics or (c) mmu-miR-33 or (d) mmu-miR-33* (left panels). Western blotting of lysates from mouse peritoneal macrophages treated with control mimics or (c) mmu-miR-33 or (d) mmu-miR-33* (right panels). ( e, f ) mRNA levels of Atg5 , Lamp1 and Prkaa1 in peritoneal macrophages transfected with ( e ) mmu-miR-33 or anti-miR33 or ( f ) mmu-miR-33* or anti-miR-33*. ( g ) qPCR analysis of miR-33 and miR-33* target gene mRNA expression in wild type (WT) and Mir33 −/− BMDMs. ( h ) Quantification of miR-33 and miR-33* gene targets in GFP + (infected) or GFP − (uninfected) alveolar macrophages isolated from mice infected with a GFP-expressing H37Rv Mtb strain for 2 weeks. * P ≤0.1, ** P ≤0.05, *** P ≤0.005 (Student’s t-test ( a–d , g , h ), one-way ANOVA ( e, f )). Data are from one experiment representative of 3 ( a–f; mean ± s.e.m) or 2 ( g; mean ± s.e.m) independent experiments. Data are from 3 mice ( h ; mean ± s.e.m).

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: Regulation of human and mouse autophagy gene targets by miR-33 and miR-33* ( a–b ) 3’UTR-luciferase reporter activity of human autophagy gene targets in HEK293 cells transfected with control (ctrl) mimics or (a) hsa-miR-33a or (b) hsa-miR-33a* (left panels). Western blotting of lysates from human THP-1 macrophages treated with control (ctrl) mimics or (a) hsa-miR-33a or (b) hsa-miR-33a* (right panels)., HSP90 is shown as an internal control (right). ( c–d ) 3’UTR-luciferase reporter activity of mouse autophagy gene targets in HEK293 cells transfected with control mimics or (c) mmu-miR-33 or (d) mmu-miR-33* (left panels). Western blotting of lysates from mouse peritoneal macrophages treated with control mimics or (c) mmu-miR-33 or (d) mmu-miR-33* (right panels). ( e, f ) mRNA levels of Atg5 , Lamp1 and Prkaa1 in peritoneal macrophages transfected with ( e ) mmu-miR-33 or anti-miR33 or ( f ) mmu-miR-33* or anti-miR-33*. ( g ) qPCR analysis of miR-33 and miR-33* target gene mRNA expression in wild type (WT) and Mir33 −/− BMDMs. ( h ) Quantification of miR-33 and miR-33* gene targets in GFP + (infected) or GFP − (uninfected) alveolar macrophages isolated from mice infected with a GFP-expressing H37Rv Mtb strain for 2 weeks. * P ≤0.1, ** P ≤0.05, *** P ≤0.005 (Student’s t-test ( a–d , g , h ), one-way ANOVA ( e, f )). Data are from one experiment representative of 3 ( a–f; mean ± s.e.m) or 2 ( g; mean ± s.e.m) independent experiments. Data are from 3 mice ( h ; mean ± s.e.m).

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Infection, Isolation, Mouse Assay

    miR-33 and miR-33* repress AMPKα and downstream transcription factors controlling autophagy and lysosomal gene programs ( a ) Western blotting of lysates from peritoneal macrophages treated with anti-miR-33 or anti-miR-33* or control (ctrl) miR in the presence or absence of the AMPK inhibitor compound C (Ampk i , 5 μM). ( b, c ) mRNA levels of Foxo3 , Tcfeb and their downstream transcriptional targets in peritoneal macrophages transfected with ( b ) miR-33 and ( c ) miR-33* mimics or inhibitors as indicated. ( d ) Foxo3 and ( e ) Tcfeb mRNA in wild-type (WT) or Ampk −/− BMDMs treated with mmu-miR-33, -33*, or Ctrl mimic. ( f ) Immunofluorescence (IF) imaging of FOXO3a and TFEB in green, F-actin (red) and DAPI (blue) in wild-type (WT) and Mir33 −/− BMDMs. ( g ) Quantification of Foxo3 , Tcfeb and their downstream transcriptional targets (right) in WT and Mir33 −/− BMDMs. ( h ) Quantification of Foxo3 , Tcfeb and their downstream transcriptional targets in GFP + (infected) or GFP − (uninfected) alveolar macrophages isolated from mice infected with a GFP-expressing H37Rv Mtb strain, 2 weeks post-infection. * P ≤0.1, ** P ≤0.05, *** P ≤0.005 (Student’s t-test ( b – c, g, h ), one-way ANOVA ( d , e )). Data are from one experiment representative of 3 ( (b, c, g; mean ± s.e.m)) or 2 (( a , f ) independent experiments. Data are from 2 experiments ( d , e; mean ± s.e.m). Data are from 3 mice ( h ; mean ± s.e.m).

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: miR-33 and miR-33* repress AMPKα and downstream transcription factors controlling autophagy and lysosomal gene programs ( a ) Western blotting of lysates from peritoneal macrophages treated with anti-miR-33 or anti-miR-33* or control (ctrl) miR in the presence or absence of the AMPK inhibitor compound C (Ampk i , 5 μM). ( b, c ) mRNA levels of Foxo3 , Tcfeb and their downstream transcriptional targets in peritoneal macrophages transfected with ( b ) miR-33 and ( c ) miR-33* mimics or inhibitors as indicated. ( d ) Foxo3 and ( e ) Tcfeb mRNA in wild-type (WT) or Ampk −/− BMDMs treated with mmu-miR-33, -33*, or Ctrl mimic. ( f ) Immunofluorescence (IF) imaging of FOXO3a and TFEB in green, F-actin (red) and DAPI (blue) in wild-type (WT) and Mir33 −/− BMDMs. ( g ) Quantification of Foxo3 , Tcfeb and their downstream transcriptional targets (right) in WT and Mir33 −/− BMDMs. ( h ) Quantification of Foxo3 , Tcfeb and their downstream transcriptional targets in GFP + (infected) or GFP − (uninfected) alveolar macrophages isolated from mice infected with a GFP-expressing H37Rv Mtb strain, 2 weeks post-infection. * P ≤0.1, ** P ≤0.05, *** P ≤0.005 (Student’s t-test ( b – c, g, h ), one-way ANOVA ( d , e )). Data are from one experiment representative of 3 ( (b, c, g; mean ± s.e.m)) or 2 (( a , f ) independent experiments. Data are from 2 experiments ( d , e; mean ± s.e.m). Data are from 3 mice ( h ; mean ± s.e.m).

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Western Blot, Transfection, Immunofluorescence, Imaging, Infection, Isolation, Mouse Assay, Expressing

    Silencing of miR-33 and miR-33* enhances Mtb targeting by the autophagy machinery ( a ) Immunofluorescence (IF) imaging of peritoneal macrophages infected with GFP-tagged H37Rv Mtb (green), p62 (red) and DAPI (blue) for 24 h and treated with control (ctrl) miR, anti-miR33 or anti-miR33*, or both. Quantification of p62 co-localization with Mtb shown at right. ( b ) IF imaging of peritoneal macrophages infected with GFP-tagged LC3 (green), H37Rv Mtb (red) and DAPI (blue) for 24h and treated with control (ctrl) miR, anti-miR33 or anti-miR33*, or both. Quantification of LC3 co-localization with Mtb shown at right. ( c – d ) IF imaging of (c) p62 (green) or (d) LC3 in wild type (WT) and Mir33 −/− macrophages infected with DsRed-H37Rv or DsRed-ΔesxA Mtb strains. Quantification is shown at right. * P ≤0.1, ** P ≤0.05 (one-way ANOVA ( a–b ), two-way ANOVA ( c–d )). Data are from one experiment representative of 2 ( a , b , d ) or 3 ( c ) independent experiments with similar findings. Scale bar = 10 μm ( a –d).

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: Silencing of miR-33 and miR-33* enhances Mtb targeting by the autophagy machinery ( a ) Immunofluorescence (IF) imaging of peritoneal macrophages infected with GFP-tagged H37Rv Mtb (green), p62 (red) and DAPI (blue) for 24 h and treated with control (ctrl) miR, anti-miR33 or anti-miR33*, or both. Quantification of p62 co-localization with Mtb shown at right. ( b ) IF imaging of peritoneal macrophages infected with GFP-tagged LC3 (green), H37Rv Mtb (red) and DAPI (blue) for 24h and treated with control (ctrl) miR, anti-miR33 or anti-miR33*, or both. Quantification of LC3 co-localization with Mtb shown at right. ( c – d ) IF imaging of (c) p62 (green) or (d) LC3 in wild type (WT) and Mir33 −/− macrophages infected with DsRed-H37Rv or DsRed-ΔesxA Mtb strains. Quantification is shown at right. * P ≤0.1, ** P ≤0.05 (one-way ANOVA ( a–b ), two-way ANOVA ( c–d )). Data are from one experiment representative of 2 ( a , b , d ) or 3 ( c ) independent experiments with similar findings. Scale bar = 10 μm ( a –d).

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Immunofluorescence, Imaging, Infection

    M. tuberculosis infection upregulates the miR-33 locus in macrophages ( a ) qPCR quantification of miR-33, miR-33*, and Srebf2 levels in peritoneal macrophages infected with H37Rv Mtb for 48 h. ( b ) qPCR quantification of S rebf2 and pre-miR-33 in BMDMs treated with lipoarabinomannan (LAM; 5 μg/mL) or trehalose dimycolate (TDM; 2.5 μg/mL) 3 h ( Srebf2 ) or 24 h (pre-miR-33) after treatment. ( c ) qPCR quantification of miR-33 and miR-33* expression in mouse embryonic fibroblasts (MEF) and peritoneal macrophages (Pmac). The mmu-miR-33 stem loop structure with the guide (miR-33; blue) and passenger (miR-33*; red) strand are shown at right. ( d ) Co-precipitation of endogenous miR-33, miR-33* or miR-27b (control) with Argonaute2 in peritoneal macrophages treated with vehicle (control) or gamma-irradiated Mtb (γ-Mtb; 10 μg/mL). ( e ) Quantification of miR-33 and miR-33* expression in GFP + (infected) or GFP − (uninfected) alveolar macrophages isolated from mice infected with a GFP-expressing H37Rv Mtb strain for 2 weeks. ( f ) Quantification of S rebf2 , miR-33 and miR-33* levels in BMDMs treated with γ-Mtb in the absence (Unstim) or presence of the NFκB inhibitor BAY11-7082 (BAY; 1 μM). Data are expressed as fold-change for the γ-Mtb treatment relative to untreated for each time point and dotted lines dileneate the threshold of control. * P ≤0.1, ** P ≤0.05 (Students’ t-test ( a , d , e ), one-way ANOVA ( b ), two-way ANOVA ( c , f )). Data are from one experiment representative of 3 independent experiments with similar results ( a , f ; mean ± s.e.m). Data are from 2 experiments ( b , c , d; mean ± s.e.m). Data are from 3 mice ( e ; mean ± s.e.m).

    Journal: Nature immunology

    Article Title: Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

    doi: 10.1038/ni.3434

    Figure Lengend Snippet: M. tuberculosis infection upregulates the miR-33 locus in macrophages ( a ) qPCR quantification of miR-33, miR-33*, and Srebf2 levels in peritoneal macrophages infected with H37Rv Mtb for 48 h. ( b ) qPCR quantification of S rebf2 and pre-miR-33 in BMDMs treated with lipoarabinomannan (LAM; 5 μg/mL) or trehalose dimycolate (TDM; 2.5 μg/mL) 3 h ( Srebf2 ) or 24 h (pre-miR-33) after treatment. ( c ) qPCR quantification of miR-33 and miR-33* expression in mouse embryonic fibroblasts (MEF) and peritoneal macrophages (Pmac). The mmu-miR-33 stem loop structure with the guide (miR-33; blue) and passenger (miR-33*; red) strand are shown at right. ( d ) Co-precipitation of endogenous miR-33, miR-33* or miR-27b (control) with Argonaute2 in peritoneal macrophages treated with vehicle (control) or gamma-irradiated Mtb (γ-Mtb; 10 μg/mL). ( e ) Quantification of miR-33 and miR-33* expression in GFP + (infected) or GFP − (uninfected) alveolar macrophages isolated from mice infected with a GFP-expressing H37Rv Mtb strain for 2 weeks. ( f ) Quantification of S rebf2 , miR-33 and miR-33* levels in BMDMs treated with γ-Mtb in the absence (Unstim) or presence of the NFκB inhibitor BAY11-7082 (BAY; 1 μM). Data are expressed as fold-change for the γ-Mtb treatment relative to untreated for each time point and dotted lines dileneate the threshold of control. * P ≤0.1, ** P ≤0.05 (Students’ t-test ( a , d , e ), one-way ANOVA ( b ), two-way ANOVA ( c , f )). Data are from one experiment representative of 3 independent experiments with similar results ( a , f ; mean ± s.e.m). Data are from 2 experiments ( b , c , d; mean ± s.e.m). Data are from 3 mice ( e ; mean ± s.e.m).

    Article Snippet: For treatment of macrophages with Mtb ligands, BMDMs were treated for 24h with 5 μg/mL of purified Lipoarabinomannan (LAM), 2.5 μg/mL of purified Trehalose Dimycolate (TDM) or 10 μg/mL of gamma-irradiated M. tuberculosis (γ-Mtb), strain H37Rv (BEI Resources).

    Techniques: Infection, Real-time Polymerase Chain Reaction, Laser Capture Microdissection, Expressing, Irradiation, Isolation, Mouse Assay