mycobacterium tuberculosis mtb genomic dna  (ATCC)

Journal: Science Advances

Article Title: A streamlined CRISPR-based test for tuberculosis detection directly from sputum

doi: 10.1126/sciadv.adx2067

Figure Lengend Snippet: ( A ) Cas13a detecting BCG genomic DNA spiked into sputum:TE matrix and Cas12a detecting internal control. Sp is sputum, and gBCG is genomic BCG. Units are genomes per microliter. Cas13a reporter is FAM, and Cas12a reporter is HEX. ( B ) Comparison of fresh Cas13a (left) or lyophilized Cas13a (right) assays detecting synthetic internal control (IC) and BCG genomic DNA. Synthetic IC and genomic BCG were mixed at a concentration of 10 4 and 10 3 copies per microliter, respectively. ( C ) Lateral flow readout of Cas13a detecting dual targets IS6110 + IS1081 (top) and Cas12a detecting internal control (bottom) in BCG spiked into sputum:TE matrix. Appearance of the top line is a positive readout. Faint lines are interpreted as negative. Disappearance of the bottom line is not necessary. ( D ) Range finding experiment demonstrating the dynamic range of fresh Cas13a dual-detection assay. Cas13a detecting BCG and Cas12a detecting internal control. BCG culture [left; P adj (left to right) is <0.001, <0.001, <0.001, <0.01, <0.05, n.s., n.s., and n.s.] and BCG spiked into sputum:TE [right; P adj (left to right) is <0.0001, <0.0001, <0.0001, <0.05, n.s., n.s., and n.s.]. gBCG is genomic BCG and units are genomes per microliter. All other units are colony-forming units (CFU) per milliliter. ( E ) Testing fresh Cas13a and Cas12a coupled assay against TB-negative clinical sputum samples. Numbers indicate patient number. All samples are n.s. except Sp-17, where P = 0.015. ( F ) LoD for H37Rv. LoD = 69.0 (51.0 to 86.9). ( G ) LoD for BCG. LoD = 80.5 (59.4 to 101.6). In (A), (B), (D), and (E), error bars: SD based on n = 3. (C) A single representative sample. (F) n = 20 and (G) n = 21. In (D) and (E), one-way ANOVA with Dunnett’s test was performed, and P adj values were calculated compared to NTC that contained water. a.u., arbitrary units.

Article Snippet: Quantitative Mtb genomic DNA was ordered from ATCC (catalog no. 25177DQ).

Techniques: Control, Comparison, Concentration Assay, Detection Assay

Journal: bioRxiv

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.1101/2025.06.23.660389

Figure Lengend Snippet: (a) Relative gene expression levels of Syt1 and Syt7 compared to Gapdh in BEAS-2B and PMA-differentiated THP-1 cells. (b) Genome editing efficiency and percent distribution of individual indels of Syt1 and Syt7 KO BEAS-2B cells as determined by Sanger sequencing and Interference of CRISPR Edits (ICE) . (c) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with H37Rv Mtb-infected (MOI=8) WT, Syt1 KO, or Syt7 KO BEAS-2B cells, represented as spot forming units (SFU). (d) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with WT, Syt1 KO, or Syt7 KO BEAS-2B cells in the presence of Msmeg supernatant, CFP102-9 peptide, or (e) 5-A-RU prodrug, represented as SFU. All data are plotted as mean±SEM and pooled from three independent experiments. For (c-e), the means of technical duplicates were pooled, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Article Snippet: H37Rv Mycobacterium tuberculosis (Mtb) strain, obtained from the American Type Culture Collection (ATCC), was grown in Middlebrook 7H9 Broth supplemented with Middlebrook ADC (BD), 0.05% Tween-80 (OmniPur), and 0.5% glycerol (Thermo Fisher Scientific).

Techniques: Gene Expression, Sequencing, CRISPR, Clone Assay, Cell Culture, Infection, Comparison

Journal: bioRxiv

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.1101/2025.06.23.660389

Figure Lengend Snippet: (a) Gating strategy of BEAS-2B cells infected overnight with auxotrophic strain mEmeraldRFP-AuxMtb (MOI=8) by gating on cells, excluding doublets using forward scatter properties, and selecting Live/Dead Near-IR stain negative cells. (b) Representative gate on GFP + population to indicate live BEAS-2B cells infected with mEmeraldRFP-AuxMtb. (c) Percent Mtb uptake measured as proportion of live WT, Syt1 KO, or Syt7 KO BEAS-2B cells that are GFP + . (d) Colony forming units (CFU) of H37Rv Mtb in WT, Syt1 KO, or Syt7 KO BEAS-2B cells after overnight infection (MOI=8). (e) Relative gene expression levels of Mr1 compared to Gapdh in WT, Syt1 KO, or Syt7 KO BEAS-2B cells. All data are plotted as mean±SEM. (c-d) are pooled from three independent experiments and (e) is pooled from two independent experiments. For (c-e), ordinary one-way ANOVA with Dunnett’s multiple comparisons test were used to analyze significant differences. ns=not significant (p>0.05).

Article Snippet: H37Rv Mycobacterium tuberculosis (Mtb) strain, obtained from the American Type Culture Collection (ATCC), was grown in Middlebrook 7H9 Broth supplemented with Middlebrook ADC (BD), 0.05% Tween-80 (OmniPur), and 0.5% glycerol (Thermo Fisher Scientific).

Techniques: Infection, Staining, Gene Expression

Journal: bioRxiv

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.1101/2025.06.23.660389

Figure Lengend Snippet: (a) Genome editing efficiency and percent distribution of individual indels of Syt1 and Syt7 KO THP-1 cells as determined by Sanger sequencing and ICE analysis . (b) IFN-γ release by MAIT cell clones co-cultured with H37Rv Mtb-infected (MOI=1) WT, Syt1 KO, or Syt7 KO THP-1 cells following PMA differentiation, represented as SFU. (c) IFN-γ release by MAIT cell clones co-cultured with WT, Syt1 KO, or Syt7 KO THP-1 cells following PMA differentiation in the presence of Msmeg supernatant, represented as SFU. All data are plotted as mean±SEM and pooled from three independent experiments. For (b-c), the means of technical duplicates were pooled, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Article Snippet: H37Rv Mycobacterium tuberculosis (Mtb) strain, obtained from the American Type Culture Collection (ATCC), was grown in Middlebrook 7H9 Broth supplemented with Middlebrook ADC (BD), 0.05% Tween-80 (OmniPur), and 0.5% glycerol (Thermo Fisher Scientific).

Techniques: Sequencing, Clone Assay, Cell Culture, Infection, Comparison

Journal: bioRxiv

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.1101/2025.06.23.660389

Figure Lengend Snippet: (a) Relative gene expression levels of Syt11, Esyt1, and Esyt2 compared to Gapdh in BEAS-2B cells. (b) Knockdown efficiency of Syt11, Esyt1, and Esyt2 after 48 hours of knockdown with missense (Mis) or gene-specific (KD) siRNA. (c-e) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with BEAS-2B cells following siRNA knockdown of Syt11 (c), Esyt1 (d), or Esyt2 (e). Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with exogenously added antigens (Msmeg supernatant and CFP102-9 peptide). IFN-γ release is represented as SFU. All data are plotted as mean±SEM and pooled from three independent experiments. For (c-e), the means of technical duplicates were pooled, and non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Article Snippet: H37Rv Mycobacterium tuberculosis (Mtb) strain, obtained from the American Type Culture Collection (ATCC), was grown in Middlebrook 7H9 Broth supplemented with Middlebrook ADC (BD), 0.05% Tween-80 (OmniPur), and 0.5% glycerol (Thermo Fisher Scientific).

Techniques: Gene Expression, Knockdown, Clone Assay, Cell Culture, Infection, Incubation

Journal: bioRxiv

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.1101/2025.06.23.660389

Figure Lengend Snippet: Syt1 KO and Syt7 KO were generated in the background of BEAS-2B MR1KO:tetMR1-GFP clone D4 cells. (a) Syt1 and Syt7 KO clones were verified by Sanger sequencing and analyzed using the ICE tool . (b) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with H37Rv Mtb-infected cells (MOI=8) is represented as SFU. The means of technical duplicates were pooled from three independent experiments, and non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test. (c-d) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells were incubated overnight with doxycycline and Ac-6-FP or NaOH (solvent control). Images (c) and histograms (d, left) representative of three independent experiments with pooled geometric mean fluorescence intensity (GeoMFI) (d, right) of surface MR1 and HLA-Ia expression. (e) Representative images of WT, Syt1 KO, Syt7 KO EAS-2B MR1KO:tetMR1-GFP cells incubated overnight with doxycycline and (f) measurement of area of MR1 vesicles, classified into small (1 vesicle) or large (>1 vesicle) vesicles (n=15). Each dot represents one cell. Data are pooled from three independent experiments. (g) Representative images of WT, Syt1 KO, Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells incubated overnight with doxycycline and CellLight BacMam 2.0 reagents for Rab5a and LAMP1 (yellow). (h) Percent co-localization of Rab5a (n=16) and LAMP1 (n=16) with MR1 vesicles. Each dot represents one cell. Data are pooled from four independent experiments. For (d, f, h), p-values were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test. All data are plotted as mean±SEM. All scale bars represent 10 µm.

Article Snippet: H37Rv Mycobacterium tuberculosis (Mtb) strain, obtained from the American Type Culture Collection (ATCC), was grown in Middlebrook 7H9 Broth supplemented with Middlebrook ADC (BD), 0.05% Tween-80 (OmniPur), and 0.5% glycerol (Thermo Fisher Scientific).

Techniques: Generated, Clone Assay, Sequencing, Cell Culture, Infection, Incubation, Solvent, Control, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Selective inhibition of MR1-restricted T cell activation by a novel MR1-targeting nanobody

doi: 10.1101/2025.06.11.659204

Figure Lengend Snippet: BEAS-2B cells were pre-treated with an irrelevant control nanobody (open circles), MR1Nb1 (filled circles), an IgG2a isotype control antibody (open squares), or the anti-MR1 antibody clone 26.5 (filled squares) at the indicated concentrations, incubated with either M. smegmatis supernatant (A) or CFP10 2-9 peptide (B), and then co-cultured with the MR1-restricted MAIT cell clone D416-G11 (A) or the HLA-B45-restricted classical T cell clone D466-A10 (B) overnight. BEAS-2B cells were infected with Mtb strain H37Rv at an MOI of 8 overnight before incubation with 5 μg/ml of each blocking reagent and subsequent co-culture with MAIT cell clone D416-G11 (C) or classical T cell clone D466-A10 (D). IFN-γ production was measured as spot-forming units (SFU) by ELISpot. Data are the pooled means of technical duplicates from (n=3) independent experiments and shown as mean +/-standard deviation.

Article Snippet: Mtb H37Rv obtained from ATCC was grown in Middlebrook 7H9 Broth supplemented with Middlebrook ADC (BD), 0.05% Tween-80 (OmniPur), and 0.5% glycerol (Thermo Fisher Scientific).

Techniques: Control, Incubation, Cell Culture, Infection, Blocking Assay, Co-Culture Assay, Enzyme-linked Immunospot, Standard Deviation

Journal: Chemmedchem

Article Title: Intracellular and Extracellular Efficacy of Homoisoflavone Derivatives Against Mycobacterium Tuberculosis : Progress Toward Novel Antitubercular Agents

doi: 10.1002/cmdc.202500249

Figure Lengend Snippet: Effect of homoisoflavone derivatives on the intracellular growth of Mycobacterium tuberculosis ( Mtb ) H37Rv in macrophages. RAW 264.7 macrophages were infected with Mtb H37Rv at an MOI of 1:1 (bacterium per macrophage) and treated with compounds 19 , 22, and 41 (0.8, 4, 20, and 100 μM) for 4 days. To measure intracellular bacterial growth, the cells were lysed on day 4 and plated in Middlebrook 7H10 agar. After 21 days, bacterial colonies were counted (CFU U test). The mean value of each group, significantly different from the mean value of the positive control (untreated infected macrophages, 62.0 ± 1.0 × 10 4 CFU/mL), was indicated by asterisks according to p < 0.001 (***), p < 0.01 (**) and p < 0.05 (*) ( n = 3). The calculated IC 50 (half‐maximal inhibitory concentration) of homoisoflavones on the intracellular growth of Mtb was inserted next to the graph.

Article Snippet: Two Mycobacterium tuberculosis strains, laboratory Mtb strain H37Rv (ATCC), and highly virulent M. tuberculosis strain of Beijing genotype (strain M299) isolated from TB patient in Mozambique) were kindly provided by Dr. Philip Suffys (FIOCRUZ, RJ/Brazil).

Techniques: Infection, Positive Control, Concentration Assay