Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal:

Article Title: Secretion of parasite-specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein-1 19 antigen

doi: 10.1046/j.1365-2567.1999.00763.x

Figure Lengend Snippet: Cytokine-dependent in vitro secretion by immune blood B lymphocytes of total IgG (a) and parasite-specific IgG (b). The means±SD of ‘n’ (Table 1) individual cultures are shown. Both sγ+/sγ− and sγ− B-cell populations were cultured with or without MSP119 in the presence of anti-CD40 mAb and cytokines as indicated. Antibody levels are indicated as the ratios of OD values from a given set of culture conditions with or without MSP119 (see the Materials and Methods). The dotted line indicates negative or background values (OD ratio = 1).

Article Snippet: In vitro culture of B lymphocytes CD19 + B cells were adjusted to 10 6 cells/ml, and cultured in 48-well plates (Falcon; Becton-Dickinson, Franklin Lakes, NJ), at a final volume of 0·5 ml of Iscove’s Dulbecco’s modified medium (Sigma, St Louis, MO) with 10% fetal calf serum (Hyclone, Logan, UT), and supplemented as previously described, 7 with or without antigen, mitogenic anti-CD40 mAb (clone ‘89’; 10 μg/ml; a gift from Dr J. Banchereau, Schering-Plough, Dardilly, France), and human recombinant cytokines: interleukin-2 (IL-2; a gift from Sanofi, Labège, France); IL-10 (a gift from Dr F. Brière, Schering-Plough, Dardilly, France); IL-6 (a gift from Dr F. Montero-Julian, Immunotech, Marseille, France); IL-1β (Peprotech, London, UK); and IL-4, obtained from Chinese hamster ovary-transfected cell cultures (a gift from Dr T. B. Nutman, National Institute of Allergy and Infectious Diseases, Bethesda, MD).

Techniques: In Vitro, Cell Culture

Journal:

Article Title: Secretion of parasite-specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein-1 19 antigen

doi: 10.1046/j.1365-2567.1999.00763.x

Figure Lengend Snippet: IgG secretion by total (sγ+/sγ−) blood B cells from P. falciparum-immune individuals is increased by exposition to MSP119 in the absence of exogenous cytokines. Histograms represent the means±SD of cultures derived from seven individuals (sγ− B cells) and 15 individuals (sγ+/sγ− B cells). Dots represent single cultures in each group. Open histograms and filled symbols represent total IgG production, and filled histograms and open symbols represent parasite-specific IgG. Antibody levels are indicated as OD ratios.

Article Snippet: In vitro culture of B lymphocytes CD19 + B cells were adjusted to 10 6 cells/ml, and cultured in 48-well plates (Falcon; Becton-Dickinson, Franklin Lakes, NJ), at a final volume of 0·5 ml of Iscove’s Dulbecco’s modified medium (Sigma, St Louis, MO) with 10% fetal calf serum (Hyclone, Logan, UT), and supplemented as previously described, 7 with or without antigen, mitogenic anti-CD40 mAb (clone ‘89’; 10 μg/ml; a gift from Dr J. Banchereau, Schering-Plough, Dardilly, France), and human recombinant cytokines: interleukin-2 (IL-2; a gift from Sanofi, Labège, France); IL-10 (a gift from Dr F. Brière, Schering-Plough, Dardilly, France); IL-6 (a gift from Dr F. Montero-Julian, Immunotech, Marseille, France); IL-1β (Peprotech, London, UK); and IL-4, obtained from Chinese hamster ovary-transfected cell cultures (a gift from Dr T. B. Nutman, National Institute of Allergy and Infectious Diseases, Bethesda, MD).

Techniques: Derivative Assay

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Scanning electron micrographs of P. aeruginosa incubated with cementoin, elafin, pre-elafin/trappin-2 or magainin 2 . P. aeruginosa (~1 × 10 7 in 500 μL) were incubated 2 h with the indicated peptides before being processed for scanning electron microscopy as described in Methods . CNT; control performed in the absence of peptides, PE; pre-elafin/trappin-2, Cem; cementoin, Ela; elafin, Mag; magainin 2. White arrows point to pore-like structures and white arrowheads to ghost bacterial cells.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Incubation, Electron Microscopy, Control

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Depolarization of P. aeruginosa membranes upon incubation with magainin 2, pre-elafin/trappin-2 or derived peptides . Fluorescence emission (arbitrary units) of the probe NPN inserted into the outer membrane ( top panel ) or the probe DiSC 3 inserted into the inner membrane ( bottom panel ) of P. aeruginosa upon addition of the indicated peptides. The controls were performed in phosphate buffer alone. Pre-elafin/trappin-2 and elafin were used at 20 μM, cementoin at 100 μM and magainin 2 at 10 μM. The arrow indicates the time-point for the addition of the various peptides.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Incubation, Derivative Assay, Fluorescence, Membrane

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2 . Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Confocal Microscopy, Incubation, Labeling

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Electrophoretic mobility shift assay of plasmid DNA incubated in the absence or presence of pre-elafin/trappin-2, elafin and cementoin . Plasmid pRS426 (100 ng) was incubated with the indicated ratios of peptide/DNA (w/w) for 1 h and then analyzed by agarose gel electrophoresis followed by staining with ethidium bromide. Above are representative gels from an experiment performed in triplicata. M; 1 kb DNA ladder from Invitrogen.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Electrophoretic Mobility Shift Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Attenuation of P. aeruginosa virulence factors by pre-elafin/trappin-2, elafin and cementoin

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques:

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Time course of DR6-induced apoptosis. After induction of DR6 expression by the addition of tetracycline (Tet), cells were harvested at various time points as indicated and divided into two portions. Half of the harvested cells were directly lysed and separated by SDS-PAGE, followed by Western blot analysis. The other half of the cells were used for preparation of cytosolic extracts and mitochondria-containing membrane fraction as described under “Experimental Procedures.” A, top panel shows induction of DR6 expression. Second panel shows PARP cleavage. Third, fourth, fifth, and sixth panels show activation of caspase-3, caspase-9, caspase-8, and caspase-7, respectively. The activation of caspases was determined by the formation of active forms of these caspases. This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (bottom panel). B, top panels show release of cytochrome c from mitochondria (right column) to the cytosol (left column). Second panels show translocation of Bax from cytosol (left panel) to mitochondria (right panel). Third panel show tBid formation and translocation to mitochondria. Fourth panel shows the reprobe of the second panels with anti-COX I antibody confirming the mitochondria remained intact during preparation.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Expressing, SDS Page, Western Blot, Membrane, Activation Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Overexpression of antiapoptotic protein Bcl-2 and Bcl-xL inhibited DR6-induced apoptosis. Cell lysis and fractionation were performed as described previously. A and B, after 24-h transfection, a significant amount of Bcl-2 and Bcl-xL was detected (lanes 3 and 4, top panel). Because the recombinant Bcl-2 and Bcl-xL were expressed as a FLAG-tagged protein, the recombinant Bcl-2 and Bcl-xL were detected with a slower migration rate than endogenous Bcl-2 and Bcl-xL (compare lanes 3 and 4 with lanes 1 and 2). This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (fourth panel). Second and third panels show tetracycline (Tet)-induced DR6 expression, respectively; fifth and sixth panels, cytochrome c release from mitochondria; seventh and eighth panels, Bax translocation from cytosol to mitochondria. C, top panel shows Western blot for PARP; second to sixth panels, Western blots for caspases; seventh panel, Western blot for actin to indicate relative loading of lysate samples; eighth and ninth panels, Western blots for Bid; tenth and eleventh panels were stained for mitochondrial protein COX I.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Over Expression, Lysis, Fractionation, Transfection, Recombinant, Migration, Membrane, Expressing, Translocation Assay, Western Blot, Staining