Journal: mSphere

Article Title: Identification of 4-Amino-Thieno[2,3- d ]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis

doi: 10.1128/mSphere.00606-19

Figure Lengend Snippet: 4-Amino-thieno[2,3- d ]pyrimidines deplete mycobacterial ATP levels. (A) M. tuberculosis ( Mtb ) was incubated in the presence of 1 μM CWHM-728, 1 μM CWHM-1023, or 400 nM Q203 for 24 h, the samples were heat inactivated, and ATP was quantified using the BacTiter-Glo assay ( n = 3). (B) M. smegmatis ( Msm ) was incubated in the presence of 10 μM CWHM-1023 for 12 h, and ATP was quantified using the BacTiter-Glo assay ( n = 3). In both panels, ATP levels were normalized to the number of log 10 CFU to account for differences in bacterial numbers due to differences in growth. P values were determined by one-way analysis of variance with Tukey’s posttest (A) or Student's t test (B). ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: Q203 was acquired from Enamine (catalog number EN-300-218150), and both bedaquiline (catalog number 465749185) and thioridazine (catalog number 1662504) were purchased from Sigma-Aldrich; all three compounds were tested in the MABA for comparative purposes.

Techniques: Incubation, Glo Assay

Journal: mSphere

Article Title: Identification of 4-Amino-Thieno[2,3- d ]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis

doi: 10.1128/mSphere.00606-19

Figure Lengend Snippet: Mycobacteria lacking cytochrome bd have increased sensitivity to 4-amino-thieno[2,3- d ]pyrimidines. (A to C) Either WT M. tuberculosis ( Mtb ) or M. tuberculosis Δ cydA was incubated in the presence of increasing concentrations of CWHM-728 (A), CWHM-1023 (B), or Q203 (C), and respiration and metabolism were measured using the MABA ( n = 3 replicates). (D) WT M. smegmatis ( Msm ) or M. smegmatis Δ cydA complemented with the empty vector (EV) or with a plasmid harboring cydAB was incubated in the presence of increasing concentrations of CWHM-1023, and respiration and metabolism were measured using the MABA ( n = 3 replicates). (A to D) Best-fit curves and IC 50 values ± standard deviation (S.D.) were generated using GraphPad Prism software. P values were determined by Student's t test (A to C) or one-way analysis of variance with Tukey’s posttest (D). *, P < 0.05; ****, P < 0.0001; ns, not significant.

Article Snippet: Q203 was acquired from Enamine (catalog number EN-300-218150), and both bedaquiline (catalog number 465749185) and thioridazine (catalog number 1662504) were purchased from Sigma-Aldrich; all three compounds were tested in the MABA for comparative purposes.

Techniques: Incubation, Plasmid Preparation, Standard Deviation, Generated, Software

Journal: International journal of molecular sciences

Article Title: Antibiotics Trigger Host Innate Immune Response via Microbiota-Brain Communication in C. elegans .

doi: 10.3390/ijms25168866

Figure Lengend Snippet: Figure 2. Q203 antibiotic activates detoxification and the innate immune response of C. elegans, possibly through inhibiting the electron transport chain of E. coli. (A,B) Images and bar graph indicate that cyp-14A4p::GFP expression is induced by E. coli mutants with a single-gene deletion in cyoA/B/C/D

Article Snippet: Q203 (MedChemExpress Cat# HY-101040, Monmouth Junction, NJ, USA) was solubilized in DMSO and added to OP50 E. coli or BW25113 E. coli, resulting in a final concentration of 1 μM, 20 μM, 100 μM, 500 μM, and 1 mM.

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: Antibiotics Trigger Host Innate Immune Response via Microbiota-Brain Communication in C. elegans .

doi: 10.3390/ijms25168866

Figure Lengend Snippet: Figure 3. Neural function is required for the activation of cyp-14A4 expression by ∆cyoB E. coli and Q203. (A,B) Microscopic images and bar graphs showing that UNC-13 is required for the activation of cyp-14A4 expression induced by ∆cyoB E. coli. The unc-13(e51) loss-of-function mutant was used to analyze the role of UNC-13 in mediating the bacteria-triggered cyp-14A4 response. The unc-13(e51);jefEx59[rgef-1p::unc-13];mgIs73[cyp-14A4p::GFP] strain of worms was utilized, wherein cyp-14A4p::GFP is expressed, and unc-13 is specifically expressed in neurons. The change in cyp- 14A4p::GFP fluorescence in the intestine was quantified through calculating the percentage change, normalized to the fluorescence levels observed in cyp-14A4p::GFP control animals treated with BW25113. The dashed line represents the GFP signal in the intestine. The data are expressed as the mean ± SEM. Significance was assessed using a two-way ANOVA test (* p < 0.05, *** p < 0.001), with a sample size of n > 20 for each group. The scale bar in the images corresponds to a length of 200 µm. (C,D) Microscopic images and bar graphs showing that UNC-13 is required for the activation of cyp-14A4 expression induced by 500 µM Q203. The change in cyp-14A4p::GFP fluorescence of the intestine was quantified through calculating the percentage change, normalized to the fluorescence levels observed in cyp-14A4p::GFP control animals treated with BW25113 + Solvent. The dashed line represents the GFP signal in the intestine. The data are expressed as the mean ± SEM. Significance was assessed using a two-way ANOVA test (* p < 0.05, ** p < 0.01), with a sample size of n > 20 for each group. The scale bar in the images corresponds to a length of 200 µm.

Article Snippet: Q203 (MedChemExpress Cat# HY-101040, Monmouth Junction, NJ, USA) was solubilized in DMSO and added to OP50 E. coli or BW25113 E. coli, resulting in a final concentration of 1 μM, 20 μM, 100 μM, 500 μM, and 1 mM.

Techniques: Activation Assay, Expressing, Mutagenesis, Bacteria, Fluorescence, Control, Solvent

Journal: International journal of molecular sciences

Article Title: Antibiotics Trigger Host Innate Immune Response via Microbiota-Brain Communication in C. elegans .

doi: 10.3390/ijms25168866

Figure Lengend Snippet: Figure 4. Brain-mediated activation of the innate immune response by ∆cyoB E. coli and Q203 enhances resistance to PA14. (A) Survival curve results showing that unc-13 mediates the activation of ∆cyoB E. coli-induced innate immune responses, improving the ability of animals to resist pathogenic bacterial PA14 virulence. The survival rates of the unc-13(e51) and wild-type N2 animals, either with ∆cyoB E. coli or BW25113 E. coli pre-treatment, were compared after exposure to PA14. Significance was assessed using Log-rank test (* p < 0.05, **** p < 0.0001, ns > 0.05), with a sample size of n > 90 for each group. (B) Survival curve results showing that unc-13 mediates the activation of Q203-induced innate immune responses, improving the ability of animals to resist pathogenic bacterial PA14 virulence. The survival rates of the unc-13(e51) and wild-type N2 animals, either with BW25113 + 500 µM Q203 or BW25113 + Solvent pre-treatment, were compared after exposure to PA14. Significance was assessed using a Log-rank test (** p < 0.01, **** p < 0.0001), with a sample size of n > 200 for each group.

Article Snippet: Q203 (MedChemExpress Cat# HY-101040, Monmouth Junction, NJ, USA) was solubilized in DMSO and added to OP50 E. coli or BW25113 E. coli, resulting in a final concentration of 1 μM, 20 μM, 100 μM, 500 μM, and 1 mM.

Techniques: Activation Assay, Solvent

Journal: International Journal of Molecular Sciences

Article Title: Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma

doi: 10.3390/ijms22126350

Figure Lengend Snippet: Macrophages from tumor-bearing mice are highly glycolytic. ( a ) Pancreata from Day 28 tumor-bearing orthotopically-transplanted (OT) mice were assessed for their myeloid cell populations by flow cytometry. Scatter plot of macrophages (CD45+ Lin- MHCIIlow-int CD24+ CD11b+ Ly6G- Ly6Clow F4/80+) that infiltrate the pancreas/tumor, as a percentage of total CD45+ cells, from OT (black) and age-matched sham controls (grey) (sham, n = 10; OT, n = 8, three independent experiments pooled). ( b ) OT mice were fed either KI20227 250 ppm (red) or standard rodent chow (black) beginning Day 1 of tumor cell transplantation. Representative scatter plot of the total flux on Day 26 as calculated from the BLI image shown (KI20227, n = 9; control, n = 10, two independent experiments). BLI images for all figures were acquired at 15-s exposure, and color scale set with lower limit at 3 × 10 6 and upper limit at 5 × 10 7 photons/sec. ( c ) Kaplan–Meier survival analysis of KI20227-fed mice ( n = 9) compared with standard chow-fed mice ( n = 10). ( d ) Sorted peritoneal (PEC) macrophages from OT and sham control mice were rested at 37 °C for 1 h and assessed for their glycolytic capacity in a live metabolic flux assay. Shown is a representative Seahorse trace of OT-PEC macrophages (black) compared with sham (grey) ( d ), and a bar graph of glycolytic capacity as calculated from the Seahorse trace ( n = 3 mice pooled per group, five independent experiments). ( e ) PEC macrophages from OT and sham controls were assessed at the transcript level for GLUT1, HK1, HK2, GPI, PFKB1, ALDOA, PGK, PKM2, LDHA, and HIF1α ( n = 3 mice pooled per group, three independent experiments). Data are means ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001 by unpaired student’s t-test with 95% confidence interval.

Article Snippet: The compounds KI20227 and WZB117 were custom-synthesized by GVK Biosciences.

Techniques: Flow Cytometry, Transplantation Assay, Control, Flux Assay

Journal: eLife

Article Title: Chalkophore-mediated respiratory oxidase flexibility controls M. tuberculosis virulence

doi: 10.7554/eLife.105794

Figure Lengend Snippet: ( A ) Heat map of transcripts encoding selected respiratory chain components determined by RNA sequencing of M. tuberculosis wild-type (WT) or Δ nrp treated with TTM or Q203. WT_GSE is the published dataset GSE159080 of M. tuberculosis H37Rv treated with Q203. Genes in the chalkophore cluster are boxed in red, genes encoding the cytochrome BD (CytBD) oxidase in blue, and genes encoding components of the bcc:aa3 supercomplex are in green. ( B ) RT-qPCR of the transcript encoding CydA in M. tuberculosis WT, Δ nrp , and complemented strain treated with varying TTM concentrations for 4 hr. Error bars represent the standard error of the mean (SEM). Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ****p<0.0001. ( C ) RT-qPCR of the transcript encoding CydA in M. tuberculosis WT, Δ nrp , and complemented strain treated with 20 μM TTM for 24 hr. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. **p<0.01, ***p<0.001, ****p<0.0001. ( D ) Dose-dependent effect of tetrathiomolybdate (TTM) on growth of the indicated M. tuberculosis strains at 7 d post inoculation. The dotted line indicates the starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05. Figure 1—source data 1. Raw data values for .

Article Snippet: Chemical compound, drug , Telacebec (Q203) , AbMole BioScience , M5297 , .

Techniques: RNA Sequencing, Quantitative RT-PCR

Journal: eLife

Article Title: Chalkophore-mediated respiratory oxidase flexibility controls M. tuberculosis virulence

doi: 10.7554/eLife.105794

Figure Lengend Snippet: ( A ) Schematic of the terminal respiratory oxidases of M. tuberculosis . The bcc:aa 3 oxidase is a heme-copper oxidase and cytochrome BD (CytBD) is a copper-independent heme oxidase. Both transfer electrons to oxygen. Q203 is an inhibitor of bcc:aa 3 by targeting the QcrB subunit, whereas ND-011992 targets CytBD. The two oxidases are individually dispensable due to compensation by the other oxidase, but M. tuberculosis lacking both is nonviable. The model to be tested is that copper chelation deprives the bcc:aa 3 oxidase of copper and that diisonitrile chalkophores counter this copper deprivation stress. ( B ) Liquid growth assays of the indicated strains with or without 20 µM TTM treatment. OD 600 at day 10 post-inoculation displayed. Dotted line indicates starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ****p<0.0001. ( C ) Bacterial survival of the indicated strains on agar media containing no addition, 1 mM BCS, or 40 μM TTM. Dotted line indicates lower limit of detection (LLOD). Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. **p<0.01, *p<0.05 ( D ) The copper deprivation sensitivity of M. tuberculosis Δ nrp Δ cydAB strain can be rescued with a synthetic diisonitrile chalkophore. Liquid growth assays of Δ nrp Δ cydAB with DMSO, 20 μM TTM, or 20 μM TTM with 10 μM of the diisonitrile chalkophore pictured in panel A. Error bars are SEM. ( E ) The bcc:aa 3 oxidase is the only target of copper starvation countered by diisonitrile chalkophores. Liquid growth assays of the indicated strains treated with 10 or 20 µM TTM, or DMSO vehicle control. OD 600 at day 10 post-inoculation displayed. Dotted line indicates starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ns = not significant, ****p<0.0001. ( F ) The effect of copper deprivation is masked by inhibition of QcrB subunit of bcc : aa 3 . Liquid growth assays of the indicated strains treated with Q203 (100 nM) alone or co-treated with 100 nM Q203 and 10 μM TTM. OD 600 at day 7 post-inoculation displayed. Dotted line indicates starting inoculum. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ***p<0.001, ns = not significant. Figure 2—source data 1. Raw data for .

Article Snippet: Chemical compound, drug , Telacebec (Q203) , AbMole BioScience , M5297 , .

Techniques: Control, Inhibition

Journal: eLife

Article Title: Chalkophore-mediated respiratory oxidase flexibility controls M. tuberculosis virulence

doi: 10.7554/eLife.105794

Figure Lengend Snippet: ( A ) Methylene blue decolorization assay of oxygen consumption under copper deprivation (tetrathiomolybdate, TTM) or treatment with Q203 in wild-type (WT) or Δ nrp Δ cydAB M. tuberculosis at day 0 (d0) or day 3 (d3) of incubation. Clear vials indicate oxygen consumption by respiration. ( B ) Quantitative measurement of oxygen consumption using oxygen-sensitive optical sensors. WT M. tuberculosis treated with DMSO, ND-011992, Q203, or both ND-011992 and Q203. Oxygen measurements were taken daily. Each point represents three measurements of two biological replicates. Error bars are SEM. Statistical significance between Q203 and ND-011992 +Q203 determined via two-way ANOVA with Tukey correction for multiple comparisons. ****p<0.0001. ( C ) Same assay as in panel B with WT and Δ nrp M. tuberculosis treated with DMSO or 25 μM TTM. Error bars are SEM. ( D ) Same assay as in panel B with WT, Δ nrp Δ cydAB, or Δ nrp Δ cydAB + nrp treated with 25 μM TTM. Error bars are SEM. Statistical significance between WT and Δ nrp Δ cydAB treated with 25 μM TTM determined via two-way ANOVA with Tukey correction for multiple comparisons. ****p<0.0001. ( E ) Cellular ATP levels determined by BacTiter-Glo in the indicated strains treated with DMSO, 20 or 40 μM TTM, or 100 nM Q203. [ATP] determined by standard curve determined in growth media containing the same quantities of DMSO, TTM, or Q203. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, ***p<0.001, ****p<0.0001. ( F ) Relative abundance of a CtaD-ALFA protein in M. tuberculosis of the indicated genotype treated with bathocuproinedisulfonic acid (BCS) or TTM. See for primary immunoblot data. Error bars are SEM. Statistical significance determined via two-way ANOVA with Tukey correction for multiple comparisons. ns = not significant. Figure 3—source data 1. Raw data for .

Article Snippet: Chemical compound, drug , Telacebec (Q203) , AbMole BioScience , M5297 , .

Techniques: Incubation, Western Blot

Journal: eLife

Article Title: Chalkophore-mediated respiratory oxidase flexibility controls M. tuberculosis virulence

doi: 10.7554/eLife.105794

Figure Lengend Snippet: ( A ) Methylene blue decolorization assay in WT M. tuberculosis or M. tuberculosis Δ nrp at assay start (d0) or after 3 d of incubation in a sealed tube (d3) treated with DMSO, 50 µM tetrathiomolybdate (TTM), or the combination of Q203 (400 nM) and ND-011992 (50 µM). ( B ) Copper chelation does not destabilize the CtaD protein M. tuberculosis lacking the CtaD subunit of the bcc:aa3 oxidase and complemented with a fully functional CtaD with a C-terminal ALFA epitope tag (see ) either in the wild-type background (WT) or M. tuberculosis Δ nrp (KO), which lacks diisonitrile chalkophore biosynthesis, and treated with either bathocuproinedisulfonic acid (BCS) or TTM. Full immunoblots for the ALFA tag or RpoB as a loading control are shown. Images were quantified using ImageJ software. Copper chelation synergizes with CytBD inhibition in the absence of chalkophore biosynthesis. Serial dilutions of M. tuberculosis WT,!1nrp,!1nrp +nrp strains were cultured on agar media containing TTM, ND-011992 (ND), or both. *p=0.0148 by two-way ANOVA. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , with bands labeled. Figure 3—figure supplement 1—source data 2. Original uncropped western blot files for . Figure 3—figure supplement 1—source data 3. Bacterial counts for .

Article Snippet: Chemical compound, drug , Telacebec (Q203) , AbMole BioScience , M5297 , .

Techniques: Incubation, Functional Assay, Western Blot, Control, Software, Inhibition, Cell Culture, Labeling

Journal: Nature Communications

Article Title: Targeting the Mycobacterium ulcerans cytochrome bc 1 : aa 3 for the treatment of Buruli ulcer

doi: 10.1038/s41467-018-07804-8

Figure Lengend Snippet: The cytochrome bc 1 :aa 3 is the sole respiratory terminal oxidase in classical M. ulcerans strains. a A single-nucleotide polymorphism (692G>A) converting the tryptophan-encoding codon TGG into the stop codon TAG is present in cydA of all classical M. ulceran s strains. b The cydAB operon present in classical M. ulcerans strains does not encode a functional cyt- bd . Q203-treated M. bovis BCGΔ cydAB strains complemented with the cydAB operon from classical lineage, or from Japanese ancestral lineage strains were incubated with Q203 (100 nM) in sealed tubes containing 0.001% methylene blue used as an oxygen sensor. Pictures were taken immediately after closing the tubes (day 0), and 5 days after incubation at 37 °C. Bedaquiline (1 mM) and DMSO (−) were used as controls. c Oxidative phosphorylation pathway in classical M. ulcerans strains, the molecular targets of Q203 and bedaquiline (BDQ) are shown

Article Snippet: The cultures were washed twice and resuspended in Middlebrook 7H9 medium without glycerol and incubated with Q203 (100 nM), bedaquiline (1 μM), or 1% dimethyl sulfoxide (DMSO) (solvent control).

Techniques: Functional Assay, Incubation, Phospho-proteomics

Journal: Nature Communications

Article Title: Targeting the Mycobacterium ulcerans cytochrome bc 1 : aa 3 for the treatment of Buruli ulcer

doi: 10.1038/s41467-018-07804-8

Figure Lengend Snippet: Potency of drugs targeting the cyt- bc 1 :aa 3 against M. ulcerans . a Growth inhibitory activity of Q203 and bedaquiline (BDQ) against classical M. ulcerans clinical isolates from Africa (blue symbols) and Australia (red symbols) replicating in culture broth medium using a resazurin-based assay. Data are expressed as the mean ± s.d. of triplicates for each concentration. b Growth inhibitory activity (MIC 50 ) of Q203 and BDQ against classical strains from African and Australian origin, and against ancestral strains from Japanese origin. MIC 50 values are the average of three strains, each tested in duplicate. c Growth inhibitory activity of Q203 and bedaquiline (BDQ) against the classical strain S1013 (red symbols) and the ancestral strain S1325 (blue symbols) replicating in culture broth medium using a turbidity-based assay. Data are expressed as the mean ± s.d. of triplicates for each concentration. The red dotted line shows the bottom plateau for the dose–response curve for bedaquiline and Q203 against the classical strain; the blue dotted line shows the bottom plateau of the dose–response curve for Q203 against the ancestral strain. d Bactericidal potency of Q203 against the classical strain S1013 replicating in culture broth medium. Q203 was tested at a concentration of 1, 4, and 16 nM in triplicate and repeated once. The dotted line represents the limit of detection. ** P value <0.001, using the Student's t test, between the untreated control group (1% DMSO) and Q203 at either 1, 4 or 16 nM

Article Snippet: The cultures were washed twice and resuspended in Middlebrook 7H9 medium without glycerol and incubated with Q203 (100 nM), bedaquiline (1 μM), or 1% dimethyl sulfoxide (DMSO) (solvent control).

Techniques: Activity Assay, Resazurin Assay, Concentration Assay, Control

Journal: Nature Communications

Article Title: Targeting the Mycobacterium ulcerans cytochrome bc 1 : aa 3 for the treatment of Buruli ulcer

doi: 10.1038/s41467-018-07804-8

Figure Lengend Snippet: Inhibition of cyt- bc 1 :aa 3 arrests oxidative phosphorylation in classical M. ulcerans . a Oxygen consumption rates in the classical lineage M. ulcerans strain S1013 treated with Q203 (5 nM, red squares), ND-11176 (50 nM, purple diamonds), streptomycin (5000 nM, green triangles) or DMSO (1%, black circles) was measured using the MitoXpress® Xtra-oxygen probe. Fluorescence (Ex 380 nm , Em 650 nm ) was measured over 350 min. The experiment was performed in triplicate and repeated two times. b Effect of Q203 (5 nM) or streptomycin (5000 nM) treatment on the oxygen consumption rate in the classical strain S1013 (dark grey bars) and the ancestral strain S1325. c , d Quantification of intracellular ATP levels in the classical strain S1013 ( c ) or in the ancestral strain S1325 ( d ) treated with a dose range of Q203, ND-11176 or bedaquiline (BDQ) for 24 h. Every concentration was tested in triplicate and the assay repeated once

Article Snippet: The cultures were washed twice and resuspended in Middlebrook 7H9 medium without glycerol and incubated with Q203 (100 nM), bedaquiline (1 μM), or 1% dimethyl sulfoxide (DMSO) (solvent control).

Techniques: Inhibition, Phospho-proteomics, Fluorescence, Concentration Assay

Journal: Nature Communications

Article Title: Targeting the Mycobacterium ulcerans cytochrome bc 1 : aa 3 for the treatment of Buruli ulcer

doi: 10.1038/s41467-018-07804-8

Figure Lengend Snippet: Efficacy of Q203 in the mouse footpad infection model of Buruli ulcer. a Bacterial loads were enumerated in the footpads of mice infected with M. ulcerans Cu001 after 4 and 8 weeks of daily treatment. Q203, rifampicin (Rif) and streptomycin (Str) were used at 0.5, 10 and 150 mg/kg body weight, respectively. Q203 and rifampicin were administered orally, streptomycin was given by the sub-cutaneous route. Ten mice per group and per time point were used. Data are expressed as mean ± s.d. Statistical analysis was performed using two-sided Mann–Whitney U test. b Footpad thickness was followed over time after infection with M. ulcerans S1013 on day 0. Q203 and rifampicin were administered orally three times per week at 0.5 and 10 mg/kg body weight, respectively, for 4 weeks (starting from week 5; treatment interval is boxed). Mean values with standard deviation are shown. The single cross symbol represents all 12 animals from the untreated group had to be euthanized at week 9 post infection due to severe disease progression. The double cross symbol represents all remaining eight animals from the rifampicin-treated group had to be euthanized at week 15 post infection due to unfavourable progression in the majority of the animals (5/8). c , f Appearance of infected foot at week 9 treated with the vehicle control ( c ) or with Q203 ( f ). Black arrows shows the site of infection. d – g Oedema formation and tissue necrosis in footpads from untreated control animals ( d ) and complete lack of oedema and tissue necrosis in Q203-treated animals ( g ) after completion of treatment at week 9. e – h Large extracellular clusters of solid-stained AFB embedded in the necrotic lesions in footpads of the untreated mice ( e ) and small numbers of AFB with beaded, not solid-stained appearance in the Q203-treated mice ( h ) at week 15. Scale bars: c , f : 1 cm; d , g : 80 μm; e , h : 200 μm; inlets in e , h : 10 μm

Article Snippet: The cultures were washed twice and resuspended in Middlebrook 7H9 medium without glycerol and incubated with Q203 (100 nM), bedaquiline (1 μM), or 1% dimethyl sulfoxide (DMSO) (solvent control).

Techniques: Infection, MANN-WHITNEY, Standard Deviation, Biomarker Discovery, Control, Staining