Journal: Blood

Article Title: Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model

doi: 10.1182/blood-2012-06-436022

Figure Lengend Snippet: AINs produce and secrete granules more effectively than do GINs. (A-C) Ultrastructural images of GINs (A), AINs (B), or freshly isolated PBNs (C) with electron microscope. The vesicles in GINs appeared to contain less dense and amorphous material, whereas those in AINs showed dense material or both dense and amorphous material. AINs appeared to have more primary and secondary-like granules than do GINs. Arrows indicate examples of vesicles and granules. Ve indicates vesicle; Pg, primary granule; and Sg, secondary granule. (D) NE activity challenged by fMLP stimuli. CB indicates cytochalasin B. (E) AINs had a greater production and secretion of the cleaved 38-kDa product of MPO upon E coli stimuli. Images at the top of the horizontal line were derived from 8% gel, whereas images under the horizontal line from 12% gel. The band intensities of Actin detected in 12% gel are similar to those in 8% gel (data not shown). (F) Effective secretion of lactoferrin by AINs versus GINs upon E coli stimuli. To make the sample orders in the medium section match those in the lysate section, the positions between nonstimulated and stimulated samples in the medium section have been switched, as indicated by a vertical line. (G) Greater production and degranulation of LL37 by AINs versus GINs. (H) Increased abundance of intracellular MMP-9 upon E coli stimuli but insufficient degranulation in both GINs and AINs. To make the sample orders in the medium section match those in the lysate section, the positions between nonstimulated and stimulated samples in the medium section have been switched, as indicated by a vertical line. In addition, the samples loaded in lysate and medium sections in panels E through H were originally separated by a molecular weight (MW) marker. This MW marker was deleted so that a blank space is shown between lysate and medium samples.

Article Snippet: Phagocytotic and bacterial killing assays Each 1 × 10 5 to 2 × 10 6 of freshly purified PBNs, mouse peripheral blood neutrophils (MPBNs), cyclophosphamide (CPA)–neutropenic mice PB neutrophils (C-MPBNs), G-CSF–induced neutrophils (GINs), and Am80-induced neutrophils (AINs) were suspended with 500 μL of DMEM with 10% FBS and incubated with log-phase Escherichia coli DH5α (ATCC), or Staphylococcus aureus (ATCC) at an MOI of 5 or 10 at 37°C for up to 60 minutes, whereas bacteria in the absence of neutrophils were used to determine growth.

Techniques: Isolation, Microscopy, Activity Assay, Derivative Assay, Molecular Weight, Marker

Journal: Blood

Article Title: Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model

doi: 10.1182/blood-2012-06-436022

Figure Lengend Snippet: Both unsorted and sorted AINs display a higher capacity for clearance of bacteria than do unsorted and sorted GINs. (A) Morphology of GINs and AINs before and after sorting with anti-CD15 antibody. Arrows indicate examples of monocytes. (B) Morphology of freshly isolated PBNs. (C-D) Quantification of monocytes (C) and band/segmented neutrophils (D) in panels A and B. (E-F) Comparison of the effect of unsorted and sorted GINs and AINs on the clearance of intracellular bacteria. Clearance efficiency was determined from the numbers of viable bacteria recovered from the intracellular compartment after infection. E coli DH5α at an MOI of 100 were used to infect sorted GINs, sorted AINs, and PBNs (F). (G) In situ labeling of bacteria with anti-OmpA antibodies in unsorted GINs and AINs.

Article Snippet: Phagocytotic and bacterial killing assays Each 1 × 10 5 to 2 × 10 6 of freshly purified PBNs, mouse peripheral blood neutrophils (MPBNs), cyclophosphamide (CPA)–neutropenic mice PB neutrophils (C-MPBNs), G-CSF–induced neutrophils (GINs), and Am80-induced neutrophils (AINs) were suspended with 500 μL of DMEM with 10% FBS and incubated with log-phase Escherichia coli DH5α (ATCC), or Staphylococcus aureus (ATCC) at an MOI of 5 or 10 at 37°C for up to 60 minutes, whereas bacteria in the absence of neutrophils were used to determine growth.

Techniques: Bacteria, Isolation, Comparison, Infection, In Situ, Labeling

Journal: Blood

Article Title: Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model

doi: 10.1182/blood-2012-06-436022

Figure Lengend Snippet: AINs possess significantly higher phagocytotic and bactericidal activities than do GINs. (A) Phagocytotic and bactericidal activities against E coli infection were determined by the number of extracellular bacteria, phagocytized bacteria, recovered intracellular bacteria, and killed bacteria. There was a 1.26- ± 0.01-fold increase in bacterial numbers over the 45 minutes of the experiment (*GINs versus AINs, P < .03 at least). (B) Confocal fluorescence microscopy of surviving and dead bacteria in GINs, AINs and PBNs. White arrow indicates surviving (green) or dead bacteria (red), whereas black arrow indicates example of neutrophil nucleus stained in red simultaneously by PI fluorescent dye. Representative confocal fluorescence images of bacteria were reproduced at 60× magnification. (C) Quantification of both extracellular bacteria after infection and in situ killed bacteria in panel B (*GINs versus AINs or PBNs, P < .04 at least). (D-E) Similar to panels A and B, phagocytic and bactericidal activities against S aureus infection were determined in GINs, AINs, and PBNs (*GINs versus AINs, P < .04 at least). (F-H) Ultrastructural images of bacterial infection of GINs (F), AINs (G), and PBNs (H) with electron microscope. Lb indicates living bacteria; Db, suggestive of dead bacteria; Pg, primary granule; and Sg, secondary granule.

Article Snippet: Phagocytotic and bacterial killing assays Each 1 × 10 5 to 2 × 10 6 of freshly purified PBNs, mouse peripheral blood neutrophils (MPBNs), cyclophosphamide (CPA)–neutropenic mice PB neutrophils (C-MPBNs), G-CSF–induced neutrophils (GINs), and Am80-induced neutrophils (AINs) were suspended with 500 μL of DMEM with 10% FBS and incubated with log-phase Escherichia coli DH5α (ATCC), or Staphylococcus aureus (ATCC) at an MOI of 5 or 10 at 37°C for up to 60 minutes, whereas bacteria in the absence of neutrophils were used to determine growth.

Techniques: Infection, Bacteria, Fluorescence, Microscopy, Staining, In Situ

Journal: Blood

Article Title: Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model

doi: 10.1182/blood-2012-06-436022

Figure Lengend Snippet: Anti-CD18 antibody neutralizes Am80-enhanced neutrophil bactericidal activities. (A) Effects of anti-CD18 antibody on neutralization of AINs, GINs, and PBNs phagocytic and bactericidal activities against E coli (**GINs versus AINs without Abs, P < .037 at least; *AINs versus AINs+Abs, P < .036 at least; GINs versus GINs+Abs, P < .006 at least; PBNs versus PBNs+Abs, P < .007 at least). Abs indicates antibodies. (B) In situ bacterial killing imaged by confocal microscopy. White arrow indicates surviving (green) or dead bacteria (red), whereas black arrow indicates example of neutrophil nucleus stained in red simultaneously by PI fluorescent dye. (C) Quantification of both extracellular bacteria after infection and in situ killed bacteria in panel B (**GINs versus AINs or PBNs, P < .006 at least; *AINs+IgG versus AINs+Abs, P < .008; PBNs+IgG versus PBNs+Abs, P < .03). (D-E) Effects of anti-CD18 antibody on neutralization of AINs, GINs, and PBNs phagocytic and bactericidal activities against S aureus (**GINs versus AINs or PBNs, P < .03 at least; *AINs+IgG versus AINs+Abs, P < .007 at least; PBNs+IgG versus PBNs+Abs, P < .02 at least).

Article Snippet: Phagocytotic and bacterial killing assays Each 1 × 10 5 to 2 × 10 6 of freshly purified PBNs, mouse peripheral blood neutrophils (MPBNs), cyclophosphamide (CPA)–neutropenic mice PB neutrophils (C-MPBNs), G-CSF–induced neutrophils (GINs), and Am80-induced neutrophils (AINs) were suspended with 500 μL of DMEM with 10% FBS and incubated with log-phase Escherichia coli DH5α (ATCC), or Staphylococcus aureus (ATCC) at an MOI of 5 or 10 at 37°C for up to 60 minutes, whereas bacteria in the absence of neutrophils were used to determine growth.

Techniques: Neutralization, In Situ, Confocal Microscopy, Bacteria, Staining, Infection

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: Effects of PIX on the antimicrobial activity of RFP against Gram-negative bacteria. (A) Combinational antimicrobial effects between PIX and RFP against K. pneumoniae ATCC 700603, A. baumannii ATCC 19606, P. aeruginosa PAO1 and E. coli ATCC 25922 by checkerboard assay. The MICs of RFP for four type strains are 64, 4, 64 and 32 μg/mL, respectively (B) The time-growth curve of four type strains treated with PIX and RFP alone or in combination (16 μg/mL + 1/2 ×MIC for ATCC 700603; 32 μg/mL + 1/8 ×MIC for ATCC 19606; 16 μg/mL + 1/2 ×MIC for PAO1; 16 μg/mL + 1/4 ×MIC for ATCC 25922). (C) Viable cell counting of bacteria after exposure to PIX (32 μg/mL) and RFP (1/4 × MIC or 1/2 × MIC) alone or in combination for 16 h. Data are presented as mean ± SD and the significances were determined by one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Representative CLSM images of (D) E. coli ATCC 25922 and (E) K. pneumoniae ATCC 700603 treated with PIX (32 μg/mL) and RFP (1/4 × MIC) alone or in combination and stained with SYTO9/PI to detect cell viability. Scale bar: 20 μm. (F) Resistance development during serial passaging of bacterial strains in the presence of RFP alone or RFP coupled with PIX (32 μg/mL).

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques: Activity Assay, Bacteria, Cell Counting, Staining, Passaging

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: PIX enhance the antimicrobial activity of RFP against multidrug-resistant strains

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques: Activity Assay

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: The interaction of PIX on bacterial cell outer membranes. (A) Outer membrane permeability was evaluated by NPN after treatment with either increasing concentrations of PIX or SPR741 (positive control, 16 μg/mL). (B) Effects of exogenous Mg 2+ (10 mM) and EDTA (1.25 mM) on the synergy between PIX and RFP against E. coli ATCC 25922. The top panel shared the same image as the rightmost panel of Fig. 1A due to the same treatment. (C) AFM and (D) TEM images of E. coli ATCC 25922 and K. pneumoniae ATCC 700603 incubated with PIX (32 μg/mL) or 0.1% DMSO (control). Red triangles indicate bacteria forming extracellular vesicles.

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques: Membrane, Permeability, Positive Control, Incubation, Control, Bacteria

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: Effect of PIX on bacterial redox homeostasis. (A) K. pneumoniae ATCC 700603, A. baumannii ATCC 19606, P. aeruginosa PAO1 and E. coli ATCC 25922 were treated with varying concentrations of PIX or positive control (PB, polymyxins B, 16 μg/mL), and the ROS level was measured by DCFH-DA. (B) DCFH-DA staining of E. coli ATCC 25922 visualized by CLSM. (C) RNS levels of bacteria treated with PIX or PB (16 μg/mL) were quantified by DAF-FM DA. (D) Intracellular ATP level detection in four type strains treated with varying concentrations of PIX (8-32 μg/mL). (E) Representative CLSM images of E. coli ATCC 25922 and K. pneumoniae ATCC 700603 following treatment with PIX (32 μg/mL) or CIP (10 × MIC). Scale bar: 20 μm. (F) Checkerboard assays of PIX and RFP against E. coli ATCC 25922 and K. pneumoniae ATCC 700603 under anaerobic conditions. (G) The antimicrobial susceptibility of GSH and L-Cys against E. coli ATCC 25922. (H) The growth of E. coli ATCC 25922 supplemented with either PIX (32 μg/mL), RFP (1/4 × MIC), GSH (1 mg/mL) or L-Cys (0.5 mg/mL). (I) E. coli ATCC 25922 was treated with a combination of PIX and RFP supplemented with or without antioxidants (GSH, 1 mg/mL or L-Cys, 0.5 mg/mL), and the cultures were plated after incubation for 6 h. (J) The SOS-associated gene recA expression of E. coli ATCC 25922 in the presence of PIX (16 μg/mL) was determined by qRT-PCR.

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques: Positive Control, Staining, Bacteria, Incubation, Expressing, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: The effect of PIX on bacterial PMF. (A) Detection of the intracellular pH of E. coli ATCC 25922 by monitoring the fluorescence intensity of BCECF-AM. (B) The growth of E. coli in pH-adjusted broth. (C) Decreased antibacterial activity of the combination of RFP and PIX in H + -reduced medium.

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques: Fluorescence, Activity Assay

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: Transcriptome alterations in E. coli ATCC 25922 treated with PIX. (A) KEGG enrichment analysis of the DEGs in E. coli after exposure to PIX (16 μg/mL) for 1 h. (B) Scheme of genes involved in flagellum assembly, quoted from the KEGG database. Yellow represents genes affected by PIX.

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques:

Journal: Microbiology Spectrum

Article Title: Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

doi: 10.1128/spectrum.02114-22

Figure Lengend Snippet: In vivo antimicrobial efficacy of combination therapy. The combination of PIX and RFP increased the survival rate of mice infected with (A) E. coli ATCC 25922 or (E) XDR E. coli Y9395 during 7 days postinfection in a peritonitis-sepsis model. The related abscess area (B and F), bacterial load quantification (C and G) and H&E staining of the abscess (D and H) are shown. Scales, 200 μm. Untreated (saline); RFP (20 mg/kg); PIX + RFP (30 + 20 mg/kg).

Article Snippet: Mid-log growth phase E. coli ATCC 25922 was diluted in MH broth to ~1 × 10 6 CFU/mL in the presence of 16 μg/mL PIX or 0.1% DMSO (control).

Techniques: In Vivo, Infection, Staining, Saline

Journal: Frontiers in Microbiology

Article Title: Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

doi: 10.3389/fmicb.2018.00494

Figure Lengend Snippet: The Mycobacterium tuberculosis (Mtb) RpoB H526D mutant strain exhibits slow growth rate and altered colony morphology. (A) Growth kinetics of Mtb strains in supplemented Middlebrook 7H9 broth. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (B) Mtb cultures were grown on Middlebrook 7H10 agar and colonies were photographed 32 days post-inoculation. Colonies of the RpoB H526D mutant are smaller, more translucent, and less regular than those of the parental strain.

Article Snippet: Briefly, mid-log-phase bacteria growing in supplemented Middlebrook 7H9 broth or 14-day nutrient-starved bacteria were fixed with 3% formaldehyde, 2.0% glutaraldehyde, 80 mM Sorenson’s phosphate, and 4 mM MgCl2, pH 7.2.

Techniques: Mutagenesis

Journal: Frontiers in Microbiology

Article Title: Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

doi: 10.3389/fmicb.2018.00494

Figure Lengend Snippet: Mycobacterium tuberculosis RpoB mutation H526D is associated with altered bacillary length and cell wall thickness. The bacillary length (A) and cell wall thickness (B) of the RpoB H526D mutant and its parental strain were evaluated by transmission electron microscopy (TEM) during mid-log-growth-phase (7H9) and after exposure to nutrient starvation for 14 days (14NS). Bars, (A) 2 μm and (B) 100 nm. Dot plot graphs of (C) bacillary length (μm) and (D) cell wall thickness (nm). Each dot represents a measurement for a single bacillus. ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, ns: non-significant.

Article Snippet: Briefly, mid-log-phase bacteria growing in supplemented Middlebrook 7H9 broth or 14-day nutrient-starved bacteria were fixed with 3% formaldehyde, 2.0% glutaraldehyde, 80 mM Sorenson’s phosphate, and 4 mM MgCl2, pH 7.2.

Techniques: Mutagenesis, Transmission Assay, Electron Microscopy

Journal: Frontiers in Microbiology

Article Title: Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

doi: 10.3389/fmicb.2018.00494

Figure Lengend Snippet: RpoB mutation H526D alters bacterial cellular metabolic activity. The RpoB H526D mutant and parental strains were grown to mid-log-phase in nutrient-rich broth (7H9) or were starved of nutrients for 14 days (14NS) prior to incubation with AlamarBlue for 18 h. The fluorescence signal was normalized based on bacterial density. RFI, relative fluorescence intensity. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Briefly, mid-log-phase bacteria growing in supplemented Middlebrook 7H9 broth or 14-day nutrient-starved bacteria were fixed with 3% formaldehyde, 2.0% glutaraldehyde, 80 mM Sorenson’s phosphate, and 4 mM MgCl2, pH 7.2.

Techniques: Mutagenesis, Activity Assay, Incubation, Fluorescence