Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 1. Comparison of the percentage of apop- tosis and necrosis of U937 macrophages infected with M. tuberculosis H37Rv and H37Ra. The MOI used was 10.

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Comparison, Infection

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 3. Alveolar epithelial cell apoptosis after M. tuberculosis H37Rv infection. A549 cells were either uninfected (A and B) or infected (C and D) with M. tuberculosis (MOI 10) and treated with staurosporine as described in Experimental procedures. Microscopic examination of staurosporine- treated cells indicated that: (A) staurosporine-treated uninfected A549 cells underwent nuclear morphological changes typical of apoptosis as demonstrated by the TUNEL assay (B); M. tuberculosis infection before adding staurosporine suppressed staurosporine-induced apoptosis in A549 cells (C and D).

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Infection, TUNEL Assay

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 6. A and B. Western blot analysis of bad and bcl-2 proteins in macrophage and alveolar epithelial cells. U937 and A549 cells were infected with M. tuberculosis H37Rv at an MOI 1:10 or cultured with medium alone for 48 h. Concentrated cell lysate-free supernatants were subjected to Western blotting (see Experimental procedures). Line 1, A549 uninfected control; line 2, infected A549; line 3, U937 uninfected control; line 4, infected U937.

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Western Blot, Infection, Cell Culture, Control

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

doi: 10.1046/j.1462-5822.2003.00312.x

Figure Lengend Snippet: Fig. 7. Effect of bacterial growth inhibition by rifampicin (protein syn- thesis inhibitor) on apoptosis levels of macrophages (A) and alveolar epithelial cells (B) after M. tuberculosis H37Rv infection. U937 mac- rophages as well as A549 alveolar epithelial cells were infected with M. tuberculosis virulent strain with an MOI of 10. To one part of the cell cultures, rifampicin was added 24 h after infection at a concen- tration 0.1 mg ml-1. Apoptosis response was assessed after 3 and 5 days by Cell Death Detection ELISA. Percentage of apoptosis was calculated in infected; infected/rifampicin-treated macrophages and alveolar epithelial cell populations in comparison with uninfected; uninfected/rifampicin-treated monolayers. *Statistically significant dif- ference between groups (P < 0.05).

Article Snippet: Bacterial strains and growth media Mycobacterium tuberculosis strains H37Rv and H37Ra were obtained from the American Type Culture Collection (ATCC) and were cultured on Middlebrook 7H10 agar for 20 days.

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: BMC Biochemistry

Article Title: Sustained activation of mTORC1 in macrophages increases AMPKα-dependent autophagy to maintain cellular homeostasis

doi: 10.1186/s12858-016-0069-6

Figure Lengend Snippet: Mycobacterial infection further increased autophagy in TSC1 KO BMMϕ. a Tsc1 KO macrophages have mTORC1-dependent inhibition of ULK1 at S757. BMMϕ cells from either TSC1 WT or KO BMMϕ were left uninfected or infected with M. tuberculosis strain H37Rv (TB) ( a , b , c ) or BCG ( d , e , f ) at a MOI of 10 for 24 h and lysed for immunoblotting analysis with the indicated antibodies. TSC1 WT and KO BMMϕ were treated with 100 ng/ml rapamycin ( b , d ) or with 100 nM bafilomycin A1 ( e , f ) for 3 h and then lysed for Western blot analysis. c , f Densitometric quantification of LC3-II ( n = 3) was normalized to total Actin in c and e . The arrow indicates LC3B-II. These experiments were repeated three times. BafA1, bafilomycin A1; BCG, Bacillus Calmette - Guérin ; Rapa, rapamycin; TB, M. tuberculosis strain H37Rv, WT, Wild type; KO, knockout; UN, uninfected. *, p < 0.05; **, p <0.01

Article Snippet: Virulent M. tuberculosis H37Rv (ATCC, 25618D-2) was purchased from ATCC.

Techniques: Infection, Inhibition, Western Blot, Knock-Out

Journal: BMC Biochemistry

Article Title: Sustained activation of mTORC1 in macrophages increases AMPKα-dependent autophagy to maintain cellular homeostasis

doi: 10.1186/s12858-016-0069-6

Figure Lengend Snippet: Infection of TSC1 f/f LysM-Cre + mice with M. tuberculosis resulted in lower mycobacterial counts at the time of the arrival of adaptive immune cells. Both TSC1 f/f LysM-Cre + and LysM-Cre - mice were aerogenously infected with M. tuberculosis H37Rv with 200-300 bacilli per lung. Bacterial burdens were measured by CFUs in the lungs ( a ) at 1, 2, 3, and 5 weeks post-aerosol infection and in the spleens ( b ) at 3 and 5 weeks post-aerosol infection ( n = 4 per time point). c Cytokine secretion from lung homogenates was determined by Luminex at 3 weeks post-aerosol infection. The data were shown as mean ± SD is shown ( n = 4). d Flow cytometry analysis of MHCII expression in TSC1 WT and KO macrophages at 3 weeks after M. tuberculosis infection is shown. MFI ± SD is shown ( n = 4). MFI = mean fluorescence intensity. *, p < 0.05

Article Snippet: Virulent M. tuberculosis H37Rv (ATCC, 25618D-2) was purchased from ATCC.

Techniques: Infection, Aerosol, Luminex, Flow Cytometry, Expressing, Fluorescence

Journal: medRxiv

Article Title: Optimizing culture-free approaches to recover high quality M. tuberculosis genomic variation

doi: 10.64898/2025.12.16.25342406

Figure Lengend Snippet: a) We conducted a serial dilution experiment in which we mixed two Mtb DNA standards representing lineage 4.1.2.1 (strain X003899) and 4.9 (strain H37ra), such that the minority strain represented 0-100% of the total DNA and then diluted Mtb DNA in human DNA, to simulate clinical samples which are overwhelmingly human DNA. We approximated the proportion of Mtb DNA found in sputum with smear microscopy grades 1+, 2+, and 3+. 7 We used hybrid capture to enrich for Mtb DNA, conducted whole genome sequencing of captured libraries on an Illumina platform. b) We additionally prospectively sampled matched Mtb diagnostic cultures and unprocessed sputum submitted to ARUP diagnostic laboratories. We performed hybrid capture and Illumina whole genome sequencing on matched samples.

Article Snippet: To measure the efficiency of hybrid capture and accuracy of variant identification in captured samples, we created mixtures of reference strains H37Ra (ATCC 25177) and X003899 (ATCC BAA-2237D-2) with well characterized genomes (representing Mtb lineages 4 and 2), in which the minor strain comprised 0, 1, 2, 5, 10, 20, and 50% of the input DNA ( ).

Techniques: Serial Dilution, Microscopy, Sequencing, Diagnostic Assay

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: BCG genomic DNA was ordered from ATCC (catalog no. 35734D-2) and quantified with dPCR.

Techniques: Control, Comparison, Concentration Assay, Detection Assay

Journal: medRxiv

Article Title: Direct detection and quantification of Mycobacterium tuberculosis from clinical samples by high-resolution melt qPCR

doi: 10.64898/2026.03.07.26347851

Figure Lengend Snippet: (A) Amplification plot of H37Rv replicates targeting the RD9 gene. (B) Corresponding melt curve showing a specific melting temperature (Tm) of ∼75°C. H37Rv DNA was serially diluted from 10 6 to 10 1 copies per reaction and analysed in triplicate. (C) Amplification plot obtained after 40 cycles for the dilutions, including the no-template control (NTC). (D) Corresponding melt curve showing a Tm of 73.7 ± 0.12°C. (E) Standard curve generated from the 10-fold serial dilutions, plotting cycle threshold (Ct) versus log copy number per reaction.

Article Snippet: Using the M. tuberculosis H37Rv (ATCC #25618DQ) reference strain, we performed asymmetric PCR with Vent (exo-) DNA polymerase (New England Biolabs).

Techniques: Amplification, Control, Generated