Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Bacteria, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Inhibition zone diameters (IZDs) caused by the AWME3 of H. illucens larvae fat against K. pneumoniae strains.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Inhibition

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Antimicrobial sensitivity of AWME3 against (A) K. pneumoniae ATCC BAA-2473, (B) K. pneumoniae KPM9, and (C) K. pneumoniae KPi1627 strains. The bacteria strains were subjected to concentrations of 1.25, 2.5, 5, 10, and 20 mg/mL of AWME3 from BSFL fat. The IZD values were measured after 12 and 24 h of incubation at 37°C. Doxycycline (DOX) used as a positive antibacterial control. All values are represented as mean ± SD, in triplicate ( n = 3). Data were analyzed by two-way ANOVA, followed by Dunnett’s Test. Data represented as significant difference as compared to positive control and p -value was ranged between. * p = 0.0138, ** p = 0.0062, **** p < 0.0001.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Bacteria, Incubation, Control, Positive Control

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Antibacterial activity measured by the MIC and MBC of AWME3 against K. pneumoniae strains.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Activity Assay, Positive Control

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Half of the inhibitory concentration (MIC 50 ) values of AWME3 and positive control against MDR K. pneumoniae strains.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Concentration Assay, Positive Control

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: The MIC 50 (IC 50 value in the figure legends) of K. pneumoniae strains treated with AWME3 from the larvae fat compared to the doxycycline (DOX) as a positive control. The MIC 50 values were calculated based on the turbidimetric assay data and compared to the positive control (DOX). The planktonic bacteria turbidity was assessed for (A,B) K. pneumoniae ATCC BAA-2473; (C,D) K. pneumoniae KPM9; (E,F) K. pneumoniae KPi1627 strains at 12 and 24 h incubation with (A,C,E) DOX; (B,D,F) AWME3. The MIC 50 values were calculated using the non-linear regression mode of Graph pad Prism 7 (Graph Pad Software Inc., San Diego, CA, United States). The (IC 50 )MIC 50 values are the average of three independent experiments ± standard deviation error mean (SEM).

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Positive Control, Turbidimetric Assay, Bacteria, Incubation, Software, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Klebsiella pneumoni ae strains resistance assessment. Resistance acquisition monitored during 16 serial passages (16 days) in the presence of sub-MIC (0.5 × MIC) of AWME3, and positive control (P/S) for (A) K. pneumoniae KPi1627, (B) K. pneumoniae KPM9, and (C) K. pneumoniae ATCC BAA-2473. The Y-axis represents the highest bacterial concentration during cell passaging. The figures are representative of three independent experiments.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Positive Control, Concentration Assay, Passaging

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Effect of AWME3 concentrations on the cell membrane perme ability of K. pneumoniae ATCC BAA-2473 strain. Planktonic bacteria sus pension at 10 8 CFU/mL was subjected to various concentrations of AW ME3 ranged from 0.5 (125 μg/mL) to 4x MIC (1,000 μg/mL), and incubated for 8 h at 37°C. REC was calculated at 0, 1, 2, 4, and 8 h based on the values of electrical conductivity. Bacteria without AWME3 treatment was considered as negative control. All values presented as the mean of three independent experiments ± SD.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Membrane, Bacteria, Incubation, Negative Control

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: Cytotoxicity of AWME3 fat on HEK-293 cell lines. Cells were treated with serial of AWME3 dilutions for 24 h. Then, viability of AWME3 treated-cells was measured using MTT assay (A,B) . Where, (A) cell lines were treated with AWME3 for 24 h (X-axis: log concentrations of AWME3 extract from 0 to 1,000 (μg/mL) and Y-axis: the percentage of normalized absorbance). (B) The of average OD 570 of the HEK-293 cell line treated with AWME3 for 24 h (Y-axis: the normalized absorbance of HEK-293 at OD 570 ), X-axis: AWME3 concentrations from 0 to 1,000 μg/mL. The IC 50 values were calculated using the non-linear regression mode of Graph pad Prism7 (Graph Pad Software Inc., San Diego, CA, United States). All Results are the mean (±SEM) from three independent experiments performed in triplicates ( n = 8). Statistical values are indicated.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: MTT Assay, Software

Journal: Frontiers in Microbiology

Article Title: Bacterial Outer Membrane Permeability Increase Underlies the Bactericidal Effect of Fatty Acids From Hermetia illucens (Black Soldier Fly) Larvae Fat Against Hypermucoviscous Isolates of Klebsiella pneumoniae

doi: 10.3389/fmicb.2022.844811

Figure Lengend Snippet: The composition of AWME3 extracted from H. illucens larvae fat under optimal conditions. (A) The percentages and the identity of the AWME3 chemical compounds detected by GC-MS analysis using the NIST-08 library; (B) The FAs profile that includes SFAs, USFAs, and FAs derivatives (FADs) statistically analyzed (*** p = 0.0002, **** p < 0.0001) by one-way ANOVA.

Article Snippet: AWME3 inhibited K. pneumoniae ATCC BAA-2473, K. pneumoniae KPM9, and K. pneumoniae KPi1627 growth at a MIC concentration 250 μg/ml.

Techniques: Gas Chromatography-Mass Spectrometry

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Plasmid Preparation, Recombinant, PCR Cloning, Expressing, Mutagenesis, Amplification, Clone Assay

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Analysis of the genetic locus affected in the A. baumannii ATCC 19606T 2010 insertion derivative. (A) Genetic map of the locus harboring the secA gene (ORF 2), which was truncated due to a transposon insertion near its 3′ end (vertical black arrow), and the ORF1 and ORF 3 flanking coding regions. The horizontal arrows represent the location and direction of transcription of predicted coding regions. The locations of the KpnI (K) and PvuII (P) restriction sites used to confirm the EZ::TN insertion site are indicated. The three long black horizontal bars represent cloned DNA regions used as probes in Southern blotting (pMU734), to complement the 2010 insertion derivative (pMU472), or to overproduce a His-tagged SecA derivative (pMU481). The short black horizontal bar flanked by primer numbers indicate the 5′-end secA region used to test transcriptional expression by qRT-PCR. (B and C) Southern blotting of KpnI-PvuII-digested total DNA isolated from the ATCC 19606T parental strain (lanes 2) and the 2010 derivative (lanes 3) probed with pMU734 (B) or the aph gene (C). (D) Western blotting of cytoplasmic proteins isolated from the ATCC 19606T parental strain (lane 1) and the 2010 derivative (lane 2) probed with anti-SecA antibodies. The white arrow indicates the truncated SecA protein produced in the 2010 insertion derivative.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Clone Assay, Southern Blot, Expressing, Quantitative RT-PCR, Isolation, Western Blot, Produced

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Transcriptional analysis of the secA gene in ATCC 19606T and 2010 cells. qRT-PCR was used to detect secA transcripts produced in the ATCC 19606T parental strain and the isogenic 2010 secA insertion mutant using the primers 3976 and 3977, which anneal to the 5′ end of the this gene, using recA expression for normalization. The error bars represent the standard error (SE) of the mean of data collected from three independent biological samples.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Quantitative RT-PCR, Produced, Mutagenesis, Expressing

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Growth of A. baumannii ATCC 19606T and 2010 under different culture conditions. (A) Growth curves of the ATCC 19606T parental strain and the 2010 derivative incubated in LB broth. The OD600 of 1-ml samples taken from a 50-ml LB broth culture was determined hourly for 8 h. (B) Growth of the ATCC 19606T parental strain, the 2010 mutant, its complemented derivative 2010.C, and the t6 BauA mutant incubated in LB broth in the absence of DIP or the presence of 100 μM or 200 μM DIP. Bacterial growth (OD600) was determined after overnight incubation (12 to 14 h) at 37°C in a shaking incubator set at 200 rpm. The error bars show standard errors (SEs) of the means from an assay done in triplicate. Inset, detection of BauA in outer membrane fractions isolated from 2010 (lane 1) and 2010.C (lane 2) cells. SDS-PAGE size-fractionated proteins were probed with anti-BauA polyclonal antibodies.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Incubation, Mutagenesis, Membrane, Isolation, SDS Page

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Cross-feeding bioassays and HPLC analysis of iron-restricted culture supernatants. (A to C) Cross-feeding bioassays were done with LB agar plates containing 200 μM DIP and seeded with 2010 (A), t6 acinetobactin uptake-deficient mutant (B), or s1 acinetobactin synthesis-deficient mutant (C) overnight-cultured bacteria. Sterile filter discs impregnated with 10 μl of 10 mM FeCl3, sterile distilled water, purified acinetobactin (Ab), or cell-free supernatants from 2010 overnight cultures, which contained a subinhibitory concentration of DIP, were deposited on the surface of each plate. Plates were incubated overnight at 37°C before results were recorded. The error bars show the standard errors (SEs) of the means from an assay performed in triplicate. (D) HPLC analysis of M9 minimal medium supernatants from ATCC 19606T (19606), 2010, and t6 overnight cultures. The elution peaks corresponding to acinetobactin (Ab) and the precursor DHBA are indicated. HPLC of sterile M9 minimal medium was used as a negative control.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Mutagenesis, Cell Culture, Bacteria, Sterility, Purification, Concentration Assay, Incubation, Negative Control

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Detection of acinetobactin transport proteins and transcripts. (A) Genetic organization of the bauDCEBA operon coding for acinetobactin transport functions. (B to D) SDS-PAGE of size-fractionated outer membrane (B) and cytoplasmic membrane (C and D) proteins, which were isolated from ATCC 19606T and 2010 cells cultured in LB broth or LB broth containing 100 μM DIP, were blotted onto nitrocellulose filters and probed with anti-BauA (B), anti-BauB (C), or anti-BauE (D) antibodies. The positions of molecular mass markers are shown on the left-hand side of each blot. (E to G) Agarose gel electrophoresis of bauA (E), bauB (F), and bauD (G) RT-PCR amplicons using as the template total RNA isolated from ATCC 19606T and 2010 cells cultured in LB broth or LB broth containing 100 μM DIP. Lane M, molecular size marker.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: SDS Page, Membrane, Isolation, Cell Culture, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Proteins differentially produced by the ATCC 19606T parental strain and the 2010 secA isogenic mutant. Proteins were grouped based on their predicted cellular functions. Numbers between parentheses indicate the number of proteins included in each functional category.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Produced, Mutagenesis, Functional Assay

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Growth of the ATCC 19606T parental strain and the 2010 SecA isogenic mutant with different salt concentrations. Experiments were done using 96-well plates containing 200 μl LB broth that were inoculated with 20 μl of overnight cultures and then incubated at 37°C in a plate-shaking incubator. The OD600 was determined hourly for 11 h. The error bars show the standard errors (SEs) of the means from an assay done in triplicate.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Mutagenesis, Incubation

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Role of SecA in virulence. G. mellonella caterpillars (n = 30) were injected with 1 × 105 bacteria of the ATCC 19606T parental strain (19606), the t6 bauA insertion derivative, or the 2010 secA insertion derivative in the absence (A) or the presence (B) of 50 μM FeCl3. Negative controls included noninjected caterpillars or caterpillars injected with comparable volumes of PBS or PBS plus 50 μM FeCl3. Caterpillar death was determined daily for 5 days during incubation at 37°C in darkness.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Injection, Bacteria, Incubation

Journal:

Article Title: Sialic Acid-Binding Immunoglobulin-Like Lectin 7 Mediates Selective Recognition of Sialylated Glycans Expressed on Campylobacter jejuni Lipooligosaccharides

doi: 10.1128/IAI.02094-05

Figure Lengend Snippet: Siglec-7-expressing NK cells and monocytes recognize C. jejuni HS:19(GM1+ GT1a+) bacteria in a sialic acid-dependent manner. Untreated or sialidase-treated HS:3 and HS:19(GM1+ GT1a+) strains were FITC labeled and incubated with human PBMCs (A) or purified NK cells (B). (A) Binding of bacteria was assessed by flow cytometry following labeling with the indicated MAbs: CD3, T cells; CD19, B cells; CD56, NK cells; CD14, monocytes. Values show the bacterial binding indices for each gated population of cells. (B) Binding of bacteria to purified NK cells from two different human donors. Open histograms show background fluorescence in the absence of added bacteria, and shaded histograms show binding signals obtained in the presence of bacteria. Values show the bacterial binding indices for each population of cells.

Article Snippet: Purified NK cells were isolated from PBMCs by negative selection using a MACS NK cell isolation kit and an autoMACS (Miltenyi Biotech, Bisley, United Kingdom).

Techniques: Expressing, Bacteria, Labeling, Incubation, Purification, Binding Assay, Flow Cytometry, Fluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host

doi: 10.3389/fcimb.2023.1085908

Figure Lengend Snippet: Analysis of bacterial pathogenic phenotypes and membrane stability. (A) Bacterial adherence to human type II alveolar epithelial cells (A549) at multiplicity of infection (MOI) of 100 for 30 min. Mean data from three independent experiments (biological replicates) is presented. Bacterial adherence was presented as percentage of CFU recovered per well relative to initial inoculum. (B) Binding of NTHi 3655 wild-type and mutants to human fibronectin. Bacterial (5×10 7 CFU) binding to human fibronectin (0.8-2.0 µg/ml) in 100 µl reactions was analysed by flow cytometry after incubation for 1 hour at 37°C. Rabbit anti-human fibronectin and FITC-conjugated swine anti-rabbit pAbs were used to detect the bacterial-bound fibronectin. Data represent mean values of three independent experiments. (C) Serum killing of NTHi 3655 wild-type and mutants. Bacterial (1.5×10 3 CFU) killing by 5% NHS was analysed by CFU count on chocolate agar. Heat-inactivated serum was included as a negative control and here no bacteria were killed (data not shown). Percentage of bacterial survival was expressed as (T t CFU/T 0 CFU)×100. T 0 represents CFU of sample plated at 0 min; and Tt represents CFU of sample plated at indicated time points. Data represent mean values of three independent experiments. (D) Outer membrane vesicles (OMVs) production among NTHi 3655 wild-type and mutants. OMVs from bacterial cultures were sucrose-density gradient purified and subjected to nanoparticle tracking analysis with a NanoSight NS300. OMV samples were diluted in PBS until 20-120 particles per frame were archived. Settings were optimized using 100nm polystyrene beads, and samples were recorded using the same settings (camera level 12, three recordings of 30 sec each). Recordings were thereafter processed using the NanoSight 3.1 software. Data represents mean values from three independent experiments. (E) Spot viability assay of bacterial survival in response to hyperosmotic environment. Bacteria that were serially diluted (10 9 to 10 4 CFU/ml) was spotted on chocolate agar without sodium chloride (NaCl) (left panel) or supplemented with 50 mM (middle panel) and 100 mM NaCl (right panel). Images were captured using ProtoCOL 3 HD (Synbiosis, UK). The assay was repeated in three independent experiments, and images from a representative experiment were shown. For panel A-D, error bars indicate standard deviations. Differences between wild-type and mutants were calculated by one-way ANOVA for panel (A, D) ; and two-way ANOVA for panel (B, C) *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.001. WT, NTHi 3655 wild-type; Δ p5 , p5 -knockout mutant (NTHi 3655Δ p5 ); Δ p5 CTD , mutant expressing P5 without CTD (NTHi 3655Δ p5 CTD ); Δ p5::p5, p5- transcomplemented NTHi (NTHi 3655Δ p5::p5 ).

Article Snippet: The human alveolar epithelial cell line A549 (ATCC CCL-185TM) (American Type Culture Collection (ATCC), Manassas, VA) was maintained in F12 medium supplemented with 2 mM L-glutamine, and 10% fetal calf serum (FCS) (Gibco, Life Technologies, Carlsbad, CA).

Techniques: Membrane, Infection, Binding Assay, Flow Cytometry, Incubation, Chocolate, Negative Control, Bacteria, Purification, Software, Viability Assay, Knock-Out, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Intracellular Fusobacterium nucleatum infection attenuates antitumor immunity in esophageal squamous cell carcinoma

doi: 10.1038/s41467-023-40987-3

Figure Lengend Snippet: a Three-dimensional visualization of α-tubulin and Fn in E109 cells. The cells were infected with Fn (MOI of 1:10) for 48 h. Scale bar: 50 μm (left). b , c Intracellular bacterial proliferation was assessed by a gentamycin protection assay. E109, Kyse150 and AKR cells were lysed at the indicated time points after Fn (MOI of 1:10) infection, and the numbers of total viable bacteria ( b ) and viable bacteria per ESCC cell ( c ) were determined by the serial dilution method. P means vs . the 24 h group. d , e FACS of PD-L1 + membrane expression in E109, Kyse150 and AKR cells after infection with Fn (MOI of 1:10) for 48 h and quantification. f , g Dichromatic IF staining of PD-L1 and Fn in E109, Kyse150 and AKR cells. The cells were infected with Fn (MOI of 1:10) for 48 h. f Representative images. Scale bar: 20 μm. g Quantification. h qRT‒PCR analysis of PD-L1 in E109, Kyse150 and AKR cells after infection with Fn (MOI of 1:1, 1:10, 1:100) for 48 h or infection with Fn (MOI of 1:10) for 24, 48 or 72 h. P means vs . the Con group. i , j Immunoblotting analysis of PD-L1 in E109, Kyse150 and AKR cells after infection with Fn ( i ) and quantification ( j ). P means vs . the Con group. k Immunoblotting analysis and quantification of PD-L1 in tumors from C57BL/6 xenografts (mean ± SD; the experiment was done once; n = 3 mice per group). l IF staining of PD-L1 or Fn in tumor tissues from C57BL/6 xenografts. Scale bar: 50 μm. Images in a and l were representative results of n = 3 independent experiments with similar results. Results in b , c , e , g , h and j were presented as n = 3 biological replicates, mean ± SD, ns means not significant. The statistical significance of results in b , c , h , j and k were determined by one-way ANOVA analysis. e and g were determined by a two-tailed unpaired t -test.

Article Snippet: The human ESCC cell line Eca109 (E109) and Kyse150 (a kind gift from professor Musheng Zeng, SYSUCC, Guangzhou, China), Jurkat cell line (ATCC TIB-152), embryonic kidney 293 T cells (ATCC CRL-3216), and mouse ESCC cell line AKR (C945, Wuhan Sunncell Biotechnology, China) were cultured in RPMI 1640 or DMEM medium (Gibco, CA, USA), respectively.

Techniques: Infection, Bacteria, Serial Dilution, Membrane, Expressing, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Intracellular Fusobacterium nucleatum infection attenuates antitumor immunity in esophageal squamous cell carcinoma

doi: 10.1038/s41467-023-40987-3

Figure Lengend Snippet: a – c Dichromatic IF staining of Fn and Fn-Dps ( a ), scale bar: 40 μm (top). Immunoblotting ( b ) and FACS ( c ) analysis of PD-L1. Cells were infected with Fn (MOI of 1:10) or treated with Fn-Dps (1 μM) for 48 h. d – f Venn diagram showing the unique and overlapping predicted transcription factors in different groups. The blue dots represent differentially expressed transcription factor genes (POU6F1, CEBPB, NR4A2 and MAFG). qRT‒PCR analysis ( f ). g Analysis of the relevance of the CD274 (PD-L1) and ATF3 genes in ESCA tissues (TCGA database, n = 173). h – j ATF3 and PD-L1 expression was analyzed by western blotting. ESCC cells were transfected with ATF3 overexpression (OE) or negative control (CTRL) vectors for 48 h ( h ). Co-IP assay ( i ). Cells were treated with Fn-Dps (1 μM) for 48 h after transfection with siRNA against ATF3 for 48 h ( j ). k , l Representative images of IHC staining ( k ) and IF staining quantification ( l ) of ATF3 or PD-L1 expression in tumors from C57BL/6 xenografts. Scale bar: 10 μm. m The interaction of Fn-Dps with ATF3 in ESCC cells after treated with Fn-Dps (1 μM) for 48 h was detected by Co-IP assay. n Analysis of CD274 WT or mutant promoter activity in 293 T cells transfected with ATF3-Flag and treated with Fn-Dps (1 μM) for 48 h. o ChIP‒qPCR analysis of the relative enrichment of ATF3 at the CD274 gene promoter in Kyse150 cells. p , q Dichromatic IF staining of ATF3 and Fn-Dps (p), scale bar: 50 μm (top). Immunoblotting of nuclear (N) and cytoplasmic (C) ( q ). Cells were treated with Fn-Dps (1 μM) for 48 h. Images in a , h , j , m , p and q were representative results of n = 3 independent experiments with similar results. Results in b , c , n , and o were presented as n = 3 biological replicates, mean ± SD. Statistical significance in b , c was determined by a two-tailed unpaired t -test. l , n and o were determined by two-way ANOVA multiple comparisons. g was determined by two-tailed nonparametric Spearman correlation analysis.

Article Snippet: The human ESCC cell line Eca109 (E109) and Kyse150 (a kind gift from professor Musheng Zeng, SYSUCC, Guangzhou, China), Jurkat cell line (ATCC TIB-152), embryonic kidney 293 T cells (ATCC CRL-3216), and mouse ESCC cell line AKR (C945, Wuhan Sunncell Biotechnology, China) were cultured in RPMI 1640 or DMEM medium (Gibco, CA, USA), respectively.

Techniques: Staining, Western Blot, Infection, Expressing, Transfection, Over Expression, Negative Control, Co-Immunoprecipitation Assay, Immunohistochemistry, Mutagenesis, Activity Assay, Two Tailed Test

Journal: Journal of Innate Immunity

Article Title: Selected β-Glucans Act as Immune-Training Agents by Improving Anti-Mycobacterial Activity in Human Macrophages: A Pilot Study

doi: 10.1159/000533873

Figure Lengend Snippet: β-Glucan immune-training agents

Article Snippet: Complete DMEM supplemented with 10% pooled human serum, 2 mM GlutaMAX, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco) was then added to the cells with immune-training agents, or medium only as negative control for 24 h. The immune-training agents used in this study were β-glucan Saccharomyces cerevisiae 10 μg/mL (WGPs, WGP dispersible; InvivoGen), β-glucan Alcaligenes faecalis 1 μg/mL (curdlan; Sigma-Aldrich), and culture supernatant from Alternaria (kind gift from Dr. Marta Romano).

Techniques: Bacteria

Journal: Journal of Innate Immunity

Article Title: Selected β-Glucans Act as Immune-Training Agents by Improving Anti-Mycobacterial Activity in Human Macrophages: A Pilot Study

doi: 10.1159/000533873

Figure Lengend Snippet: Immune training with selected β-glucans increases macrophage control of M. tuberculosis . a Schematic overview of experimental setup. Human monocytes were trained with curdlan 1 μg/mL, WGP dispersible 10 μg/mL, or Alternaria supernatant 0.01% for 1 day and then infected with H37Rv-GFP, MOI-10 on day 7. b Growth of H37Rv-GFP over time presented as fold change of total integrated green fluorescence. Mean values with SEM from 6 experiments are shown in graph. c Endpoint comparison of bacterial growth at 5 days post-infection. As has been indicated with closed symbols, two donors did not show improved bacterial control after WGP dispersible training. * p < 0.05, ns, not significant. d Phagocytosis as measured by internalized GFP-expressing bacteria (total integrated green fluorescence) at 4 h post-infection. * p < 0.05, ns, not significant.

Article Snippet: Complete DMEM supplemented with 10% pooled human serum, 2 mM GlutaMAX, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco) was then added to the cells with immune-training agents, or medium only as negative control for 24 h. The immune-training agents used in this study were β-glucan Saccharomyces cerevisiae 10 μg/mL (WGPs, WGP dispersible; InvivoGen), β-glucan Alcaligenes faecalis 1 μg/mL (curdlan; Sigma-Aldrich), and culture supernatant from Alternaria (kind gift from Dr. Marta Romano).

Techniques: Control, Infection, Fluorescence, Comparison, Expressing, Bacteria

Journal: Journal of Innate Immunity

Article Title: Selected β-Glucans Act as Immune-Training Agents by Improving Anti-Mycobacterial Activity in Human Macrophages: A Pilot Study

doi: 10.1159/000533873

Figure Lengend Snippet: Cytokine production in β-glucan trained human macrophages after secondary stimulation. Human monocytes were trained with curdlan 1 μg/mL (red), WGP dispersible 10 μg/mL (green), or Alternaria supernatant 0.01% (orange) for 1 day. After 1 week of washout and differentiation, the cells were either unstimulated or stimulated with a cocktail of LPS 10 ng/mL and Pam3Cys 20 ng/mL (LPS/Pam3), or γ-irradiated H37Rv MOI-10 (γRv). After 1 day, supernatants were collected and analyzed for cytokines. β-Glucan-trained samples were compared to their mock-trained controls (blue) from the same blood donor. a IL-6. b TNF-α. c IL-1β. d IL-10. e IL-8. f CXCL10. g CXCL9. h CXCL11. ns, not significant. * p < 0.05. n = 6.

Article Snippet: Complete DMEM supplemented with 10% pooled human serum, 2 mM GlutaMAX, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco) was then added to the cells with immune-training agents, or medium only as negative control for 24 h. The immune-training agents used in this study were β-glucan Saccharomyces cerevisiae 10 μg/mL (WGPs, WGP dispersible; InvivoGen), β-glucan Alcaligenes faecalis 1 μg/mL (curdlan; Sigma-Aldrich), and culture supernatant from Alternaria (kind gift from Dr. Marta Romano).

Techniques: Irradiation

Journal: Journal of Innate Immunity

Article Title: Selected β-Glucans Act as Immune-Training Agents by Improving Anti-Mycobacterial Activity in Human Macrophages: A Pilot Study

doi: 10.1159/000533873

Figure Lengend Snippet: IL-6 production in WGP dispersible-trained macrophages correlates to mycobacterial growth reduction. a Growth of H37Rv-GFP in WGP dispersible-trained macrophages at 120 h presented as fold change over 4 h post-infection. b IL-6 concentration in 24-h supernatants after secondary stimulation with either a cocktail of LPS 10 ng/mL and Pam3Cys 20 ng/mL (LPS/Pam3), or γ-irradiated H37Rv MOI-10 (γRv). β-Glucan-trained samples (green) were compared to their mock-trained controls (blue) from the same blood donor. Donors were divided into “responders” (open circles) and “non-responders” (closed circles) according to their ability to control growth of H37Rv, as shown in panel a . ns, not significant. * p < 0.05. n = 6.

Article Snippet: Complete DMEM supplemented with 10% pooled human serum, 2 mM GlutaMAX, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco) was then added to the cells with immune-training agents, or medium only as negative control for 24 h. The immune-training agents used in this study were β-glucan Saccharomyces cerevisiae 10 μg/mL (WGPs, WGP dispersible; InvivoGen), β-glucan Alcaligenes faecalis 1 μg/mL (curdlan; Sigma-Aldrich), and culture supernatant from Alternaria (kind gift from Dr. Marta Romano).

Techniques: Infection, Concentration Assay, Irradiation, Control

Journal: Journal of Innate Immunity

Article Title: Selected β-Glucans Act as Immune-Training Agents by Improving Anti-Mycobacterial Activity in Human Macrophages: A Pilot Study

doi: 10.1159/000533873

Figure Lengend Snippet: DNA methylation pathway analysis of macrophages trained with β-glucan. Macrophages from 7 donors, trained with curdlan ( a ), WGP dispersible ( b ), or Alternaria for 24 h ( c ), followed by 5 days washout/resting period. ∆β values of differentially methylated genes (DMGs) were analyzed using the PANTHER database. Pathways with enrichment in over 5 donors are shown in figure for each β-glucan-training agent. The size of the circle describes the number of genes from the input list present in the leading edge of the pathway genes, the color of the circle demonstrates the normalized enrichment score from the PANTHER database, and the circles’ location on the x -axis describes the total number of genes involved in the pathway that was identified in each donor. For WGP dispersible-trained cells, donors are divided into “responders” (green) and “non-responders” (orange) according to their ability to control growth of H37Rv.

Article Snippet: Complete DMEM supplemented with 10% pooled human serum, 2 mM GlutaMAX, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco) was then added to the cells with immune-training agents, or medium only as negative control for 24 h. The immune-training agents used in this study were β-glucan Saccharomyces cerevisiae 10 μg/mL (WGPs, WGP dispersible; InvivoGen), β-glucan Alcaligenes faecalis 1 μg/mL (curdlan; Sigma-Aldrich), and culture supernatant from Alternaria (kind gift from Dr. Marta Romano).

Techniques: DNA Methylation Assay, Methylation, Control