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: 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