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: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) Naive B cells purified from the spleen of Igk −/− B1-8 flox/+ mice were stimulated with NP-CGG or NP-Ficoll. ( A ) 3 H-thymidine incorporation on day 3. ( B ) IgM and IgG concentrations in the culture supernatants on day 3. ( C–E ) Igk −/− B1-8 flox/+ B cells were stimulated with none (–), NP-CGG, or NP-Ficoll and IL-1α, IL-1β, or IFNα, as indicated. ( C ) Enzyme-linked immunosorbent assay (ELISA) of IgG3 in the culture supernatant on day 3. AU, arbitrary units. ( D ) Representative flow cytometric plots on day 3 with the numbers indicating percentages of IgG3 + B cells (top). The frequencies of IgG3 + cells among the B cells (bottom). ( E ) quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of the circle Iγ3-Cµ and Aicda transcripts on day 2. Data are means ± standard deviations (SDs) of two ( B and C ), two to three ( D ), or three ( A ) biological replicates or three technical replicates ( E ). The data are representative of at least three ( A and B ) or two ( C–E ) independent experiments. *p < 0.05; ***p < 0.001; ****p < 0.0001; p values were calculated by one- ( B ) or two ( C and D )-way analysis of variance (ANOVA) with Tukey’s test. Figure 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A–C ) Igk −/− B1-8 flox/+ naive B cells were stimulated with nothing (–), NP-CGG, or NP-Ficoll in the presence of LPS, R-848, or CpG. ( A ) IgG concentration in the culture supernatants on day 3. ( B ) Representative flow cytometric plots on day 3 showing percentages of IgG + B cells. ( C ) qRT-PCR analysis of Aicda transcripts on day 2. ( D and E ) Igk −/− B1-8 flox/+ naive B cells were stimulated with NP-Ficoll and each indicated cytokine. ( D ) IgG3 in the culture supernatants on day 4 titrated by enzyme-linked immunosorbent assay (ELISA). AU, arbitrary units. ( E ) Representative flow cytometric profiles of IgG1, IgG2b, IgG2c, and IgG3 on day 3 (left) and the ratio of B cells expressing these IgG subclasses estimated from the flow cytometry data (right). Data are means of three biological replicates ( E ), means ± standard deviations (SDs) of two biological replicates ( A ), or three technical replicates ( C ). The data are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p values were calculated by two-way analysis of variance (ANOVA) with Tukey’s test ( A ). Figure 1—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Immunoblot analysis of the indicated molecules in Igk −/− B1-8 flox/+ B cells stimulated with NP-CGG or NP-Ficoll for the indicated time periods. ( B–D ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells were stimulated with NP-Ficoll and IL-1α, IL-1β, or IFNα. The B cells were labeled with CellTrace Violet (CTV) before culture in C. ( B ) Enzyme-linked immunosorbent assay (ELISA) of IgM and IgG3 in the culture supernatants on day 5. AU, arbitrary units. ( C ) Representative flow cytometric plots of the B cells on day 3 showing percentages of IgG3 + B cells (left). The frequencies of IgG3 + cells at each cell division number (right). ( D ) qRT-PCR analysis of the circle Iγ3-Cµ and Aicda transcripts on day 2. Data are means ± standard deviations (SDs) of two ( B ) or three ( C ) biological replicates or three technical replicates ( D ). The data are representative of at least three ( A and B ) or two ( C and D ) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated by unpaired multiple t -test ( B and C ). Figure 2—source data 1. Source data for . Figure 2—source data 2. Source data for . Figure 2—source data 3. Source data for . Figure 2—source data 4. Source data for . Figure 2—source data 5. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Western Blot, Labeling, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ naive B cells were cultured with medium alone (–) or NP-Ficoll. ( A ) 3 H-thymidine incorporation on day 3. ( B ) IgM concentration in the culture supernatant on day 4. ( C ) The histogram of CellTrace Violet (CTV) in cells analyzed in . ( D–G ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd − /− Igk −/− B1-8 flox/+ naive B cells were cultured with NP-Ficoll and LPS, R-848, or CpG ( D and E ) or LPS, R-848, or CpG alone ( F and G ). ( D and F ) IgM and IgG concentrations in the culture supernatants on day 3. ( E and G ) qRT-PCR analysis of Aicda transcripts on day 2. Data are means ± standard deviation (SD) of three ( A ) or two to three ( B ), and two ( D and F ) biological replicates, or three technical replicates ( E and G ). The data are representative of two independent experiments. *p < 0.05; ***p < 0.001; p values were calculated by unpaired multiple t -test ( A, B, and D ). Figure 2—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Cell Culture, Concentration Assay, Quantitative RT-PCR, Standard Deviation

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Flow cytometric analysis of spleen cells and peritoneal cavity cells of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd fl/fl mice. Gating strategy of follicular B cells (Fo B; CD19 + CD43 − CD21 + CD23 + ), marginal zone B cells (MZ B; CD19 + CD43 − CD21 high CD23 low ), B1a cells (CD19 + CD43 + CD5 + ), and B1b cells (CD19 + CD43 + CD5 − ) in the spleen is shown (left). The numbers of Fo B, MZ B, B1a, and B1b cells in spleen (middle) and peritoneal cavity (right) are plotted. ( B ) Enzyme-linked immunosorbent assay (ELISA) of anti-NP IgM, IgG1, and IgG3 in the sera of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd fl/fl mice at indicated weeks after immunization with NP-CGG in alum. AU, arbitrary units. Small horizontal bars are the means of four ( A ) or six ( B ) biological replicates. Each symbol represents an individual mouse. The data are representative of three ( A ) or two ( B ) independent experiments. *p < 0.05; ****p < 0.0001; p values were calculated by unpaired multiple t- test. Figure 3—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A–C ) Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice were immunized with NP-Ficoll. ( A ) Enzyme-linked immunosorbent assay (ELISA) of serum anti-NP IgM and IgG3 at the indicated weeks. ( B ) ELISA of serum anti-NP IgG1, IgG2b, and IgG2c at 2 weeks after immunization. ( C ) Representative flow cytometric plots of the spleen cells on day 7 after immunization. The numbers indicate the percentage of cells in each gate (left). The frequencies of IgM + , IgG2b + , and IgG3 + cells among NP-binding plasma cells (PCs; NP + CD138 + ; middle) and the numbers of such cells per 1 × 10 7 total lymphocytes (right) are plotted. ( D ) ELISA of anti-PPS3 IgM and IgG3 in the serum of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice at the indicated weeks after immunization with PPS3. Results are presented in AU, arbitrary units ( A, B, and D ). Small horizontal bars are the means of six to eight (A and B), five ( C ), and eight ( D ) biological replicates. Each symbol represents an individual mouse. The data are representative of three ( A and C ) or two ( B and D ) independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; p values were calculated by unpaired multiple ( A, C, and D ) or two-tailed unpaired Welch’s t -test ( B ). Figure 3—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Clinical Proteomics, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) B cells purified from Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi mice were labeled with CellTrace Violet (CTV) and transferred into B6 mice, which were immunized with NP-Ficoll on the next day. Spleen cells were analyzed 3 days later. ( A ) Representative flow cytometric plots of the spleen cells with the numbers indicating percentages of the cells within the neighboring gates (left). The frequency of IgG3 + cells among whole donor B cells (CD45.1 + CD19 + CD138 – ) (middle) and at each cell division number (right) are shown. ( B ) qRT-PCR analysis of the indicated transcripts in the donor B cells collected as in . ( C ) Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi B cells transduced with an empty vector (Ev) or vectors expressing AID or PKCδ were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day as in . Representative flow cytometry plots of the donor cells transduced with the vectors (CD45.1 + GFP + , gated as in ) (left) and the frequency of IgG3 + cells among the CD45.1 + GFP + cells (right) on day 3 after transfer. Small horizontal bars are the means of five ( A ) or six to nine ( C ) biological replicates. Each symbol represents an individual mouse (A, middle; C ). The symbols are the means of five biological replicates (A, right). Data are means ± standard deviations (SDs) of three technical replicates pooled from five mice ( B ). The data are representative of three ( A ) or two ( B ) independent experiments or is pooled from two independent experiments ( C ). ns, not significant (p > 0.05); **p < 0.01; ****p < 0.0001; p values were calculated by unpaired multiple t -test ( A ) or one-way analysis of variance (ANOVA) with Tukey’s test ( C ). Figure 4—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Purification, Labeling, Quantitative RT-PCR, Transduction, Plasmid Preparation, Expressing, Flow Cytometry

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Sorting strategy for donor B cells used in . The starting population was spleen cells of B6 mice transferred with Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi , CD45.1 + B cells and immunized with NP-Ficoll 3 days previously. CD45.1 + cells were enriched by a MACS system (left) and then donor B cells (CD45.1 + CD19 + CD138 − ) were further sorted by flow cytometry (right). ( B and C ) Strategy for retrovirus (Rv) transduction and the analysis of the TI-2 response (B, left). B cells collected from Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi mice immunized with NP-Ficoll 1 day previously were infected with Rv and cultured for 1 day. Some Rv-transduced cells were further cultured in medium for 6 hr and analyzed by flow cytometry. Representative data of the cells transduced with pMXs-IRES-GFP empty vector (Ev) is shown (B, right). Other Rv-transduced cells were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day. Spleen cells of recipient mice were analyzed or used for cell sorting 3 or 4 days later. Gating strategy of donor cells transduced with vectors (CD45.1 + GFP + ) and the expression profile of IgG3 are shown (C, left). After the enrichment of CD45.1 + cells by a MACS system, the vector-transduced donor B cells (CD45.1 + GFP + CD19 + CD138 – ) were sorted as shown (C, right). The data are representative of at least three independent experiments.

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Flow Cytometry, Transduction, Infection, Cell Culture, Plasmid Preparation, FACS, Expressing

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) qRT-PCR analysis of the transcripts of the indicated genes in Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi donor B cells (CD45.1 + CD19 + CD138 − ) purified as in from the recipient mice immunized with NP-Ficoll 3 days previously. Shown is the relative expression of each gene in Prkcd f/f Cd19 cre/+ cells to that in Prkcd +/+ Cd19 cre/+ cells. ( B ) qRT-PCR analysis of Batf transcripts in Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells cultured with medium alone (–) or with the indicated stimuli for 2 days. ( C ) Immunoblot analysis in Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells stimulated with NP-Ficoll for the indicated times. ( D and E ) B1-8 hi B cells transduced with knockdown vectors for luciferase (shControl) or BATF (shBATF) were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day as in , and their spleen cells were analyzed on day 4 after transfer. ( D ) qRT-PCR analysis of Aicda and Batf transcripts in the vector-transduced donor B cells (CD45.1 + GFP + CD19 + CD138 − ) collected as in . ( E ) Representative flow cytometric plots of the transduced donor cells with the numbers indicating the percentage of IgG3 + cells (left) and the frequency of the IgG3 + cells among such cells (right) gated as in . ( F and G ) Prkcd f/f Cd19 cre/+ B1-8 hi B cells transduced with Ev or vectors expressing BATF or PKCδ were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day, and their spleen cells were analyzed 3 days after transfer. ( F ) qRT-PCR analysis of Aicda transcripts in the vector-transduced donor B cells. ( G ) Representative flow cytometric plots of the transduced donor cells with the numbers indicating the percentage of IgG3 + cells (left) and the frequency of the IgG3 + cells among such cells (right). Data are means ± standard deviations (SDs) of three technical replicates ( A, B, D, and F ). Samples were pooled from five to eight mice ( D and F ). Small horizontal bars are the means of four ( E ) or eight to nine ( G ) biological replicates. Each symbol represents an individual mouse ( E and G ). The data are representative of two independent experiments ( A–F ) or are pooled from two independent experiments ( G ). ***p < 0.001; ****p < 0.0001; p values were calculated by two-tailed unpaired Student’s t -test ( E ) or one-way analysis of variance (ANOVA) with Tukey’s test ( G ). Figure 5—source data 1. Source data for . Figure 5—source data 2. Source data for . Figure 5—source data 3. Source data for . Figure 5—source data 4. Source data for . Figure 5—source data 5. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Quantitative RT-PCR, Purification, Expressing, Cell Culture, Western Blot, Transduction, Knockdown, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice were cohoused for at least 4 weeks and treated with 3 % dextran sodium sulfate (DSS) for 7 days. ( A ) Serum IgM, IgG1, IgG2b, and IgG3 against fecal bacteria after cohousing were quantified by enzyme-linked immunosorbent assay (ELISA). AU, arbitrary units. ( B ) Colony-forming unit (CFU) of aerobes and anaerobes in the blood 7 days after DSS treatment. ( C ) Kaplan–Meier survival plot of 16 ( Cd19 cre/+ Prkcd +/+ ) and 12 ( Cd19 cre/+ Prkcdf f/f ) mice at indicated days. Small horizontal bars are the means of 7 ( A ), or 15 ( Cd19 cre/+ Prkcd +/+ ) or 11 ( Cd19 cre/+ Prkcdf f/f ) in ( B ), biological replicates. Data were obtained from 16 ( Cd19 cre/+ Prkcd +/+ ) or 12 ( Cd19 cre/+ Prkcdf f/f ) mice in ( C ). Each symbol represents an individual mouse ( A and B ). The data are representative of two independent experiments ( A ) or pooled from two independent experiments ( B and C ). **p < 0.01; ***p < 0.001; p values were calculated by two-tailed unpaired Welch’s t -test ( A ), unpaired multiple t -test ( B ), or log-rank test ( C ). Figure 6—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: B-cell receptor (BCR) stimulation with a TI-2 antigen (Ag) activates PKCδ signaling and induces the expression of BATF, which works cooperatively with secondary signals induced by IL-1α/β, IFNα, or TLR ligands and drives the transcription of activation-induced cytidine deaminase (AID) to induce IgG CSR.

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Expressing, Activation Assay

Journal: mBio

Article Title: Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii

doi: 10.1128/mBio.01660-15

Figure Lengend Snippet: G. mellonella differentiates pathogenic and nonpathogenic Acinetobacter strains. (A) G. mellonella larvae were inoculated with 10 6 CFU of the strains indicated . Larvae were homogenized and bacteria were quantified immediately following infection ( t = 0) and after 4 h at 37°C ( t = 4). Data from a representative experiment are presented as the ratio of the number of CFU recovered at t = 4 to the number of CFU recovered at t = 0 (error bars, 1 standard deviation). (B) G. mellonella larvae were inoculated with 10 6 CFU of the strains indicated . Survival was monitored daily for 6 days. ****, P < 0.0001; ***, P < 0.001. ADP1, A. baylyi ADP1; 17978, A. baumannii ATCC 17978; AB5075, A. baumannii AB5075.

Article Snippet: For example, shows that Acinetobacter baylyi , a well-characterized, nonpathogenic Acinetobacter species, does not survive for 4 h within larvae, whereas the human-pathogenic isolates ATCC 17978 and AB5075 survive and grow within the larvae during the same time period.

Techniques: Bacteria, Infection, Standard Deviation

Journal:

Article Title: Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

doi: 10.1128/IAI.71.5.2365-2372.2003

Figure Lengend Snippet: Bacterial species and strains used in this study and their invasion of HUVEC monolayers

Article Snippet: Streptococcus gordonii strain CH1 (Challis) was chosen as the paradigm for most of the experiments reported here because it is virulent in the rat model of infection ( 59 ), several adhesion-deficient mutants were available for comparison ( 14 , 39 , 40 ), and it produces large amounts of the alpha-hemolysin (hydrogen peroxide) ( 3 , 4 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Description Source or reference HUVEC invasion a S. gordonii DL1 (Challis) Wild-type reference strain 52 17 ± 5 EM230 DL1 hsa :: ermAM 52 4 ± 2 OB219 DL1 sspA ′ sspB ′:: ermAM 14 20 ± 4 OB220 DL1 sspA :: ermAM 14 19 ± 6 OB235 DL1 cshA3 :: ermAM 40 2 ± 0.4 OB271 DL1 cshB2 :: ermAM 40 2 ± 1 OB277 DL1 cshA31 :: cat cshB2 :: ermAM 40 2 ± 0.6 CH1 (Challis) Wild-type reference strain 59 82 ± 15 AMS12 CH1 with 1.7-kb Hin d fragment of gtfG replaced with lacZ /Erm r 59 14 ± 0.5 M5 Wild type from human dental plaque 28 5 ± 2.5 S. sanguis L22 Wild type from human dental plaque 28 76 ± 11 L52 Wild type from human dental plaque 28 9 ± 2 L74 Wild type from human dental plaque 28 52 ± 8 L79 Wild type from human dental plaque 28 14 ± 5 133-79 Wild type from human endocarditis 28 21 ± 5 2017-78 Wild type from human endocarditis 28 25 ± 3 10556 Wild type from human endocarditis ATCC 98 ± 18 S. oralis KS32AR Wild type from dental plaque 8 9 ± 8 16532AR Wild type from dental plaque 8 3 ± 2 S. mitis NCTC 10712 Wild type from dental plaque 8 23 ± 6 OP51 Wild type from dental plaque 8 35 ± 9 S. mutans UA159 Wild type from dental plaque 60 26 ± 7 S. salivarius 13419 Wild type from human saliva ATCC 2 ± 1 E. coli HB101 Hybrid of strains K-12 and B, transformable strain 7 <0.01 Open in a separate window a Average number of intracellular bacteria (× 10 4 ) per HUVEC monolayer ± standard deviation of the mean ( n = 6). b ATCC, American Type Culture Collection.

Techniques:

Journal: Scientific Reports

Article Title: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

doi: 10.1038/s41598-021-82287-0

Figure Lengend Snippet: Design and evaluation of single guide RNAs (sgRNAs) targeting core molecular clock family members. ( a ) Genomic location of sgRNAs targeting Cry1 , Cry2 , Per1 , Per2 , and Bmal1 . Gray boxes indicate exons. Lines indicate introns. Red arrows indicate the location of sgRNAs. ( b ) Structure of sgRNA expression vectors driven by the U6 promoter (hU6). ITR, inverted terminal repeat; hSyn, human synapsin promoter; KASH, Klarsicht ANC-1 syne homology domain for nuclear membrane targeting; hGH pA, poly adenylation signal derived from human growth hormone. ( c ) Mutation efficiency determined by Surveyor cleavage assays. Red arrows indicate cleaved PCR products, indicating introduced mutations. Homo, homoduplex of PCR-amplified DNA fragments from the genomic DNA of non-transfected Neuro2a cells; Hetero, heteroduplex of PCR-amplified DNA fragments from the genomic DNA of non-transfected and sgRNA-expressing-plasmid-transfected Neuro2a cells.

Article Snippet: The membranes were blocked in Tris buffered with Tween-20 (TTBS) with 1% bovine serum albumin (BSA) and incubated overnight at 4 °C in TTBS with 0.1% BSA with the following primary antibodies: anti-mPer1 (#AB2201, Merck Millipore), anti-PER2 (#AB2202, Merck Millipore), anti-BMAL1 (#NB100-2288, Novus, Centennial, CO, USA), anti-CRY1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), anti-CRY2 (#HPA037577, Atlas Antibodies, Bromma, Sweden), and anti-β-actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA) antibodies.

Techniques: Expressing, Membrane, Derivative Assay, Mutagenesis, Amplification, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

doi: 10.1038/s41598-021-82287-0

Figure Lengend Snippet: Reduced protein levels of core clock components by genome editing with a multiplexed sgRNA-expressing cassette. ( a ) Structure of multiplexed U6 promoter-driven sgRNA expression cassettes for AAV packaging. ( b ) Representative western blots of protein extracts from Neuro2a-Cas9 cells transfected with CSAC- Crys (left), CSAC- Pers (middle), or CSAC- Bmal1 (right). Proteins from CSAC- Crys -infected cells were probed with anti-CRY1 and anti-CRY2 antibodies. Proteins from CSAC- Pers -infected cells were probed with anti-PER1 and anti-PER2 antibodies. Proteins from CSAC- Bmal1 -infected cells were probed with anti-Bmal1 antibodies, with anti-β-actin antibodies used as an internal control for total protein abundance. ( c – e ) Percentage of indel mutation induced by CSAC- Crys ( c ), CSAC- Pers ( d ), CSAC- Bmal1 ( e ), and LacZ, respectively. On-target and off-target sites of each sgRNA included in CSAC are indicated. Orange bar: frame-shift mutation; Light orange bar: non-frame-shift mutation.

Article Snippet: The membranes were blocked in Tris buffered with Tween-20 (TTBS) with 1% bovine serum albumin (BSA) and incubated overnight at 4 °C in TTBS with 0.1% BSA with the following primary antibodies: anti-mPer1 (#AB2201, Merck Millipore), anti-PER2 (#AB2202, Merck Millipore), anti-BMAL1 (#NB100-2288, Novus, Centennial, CO, USA), anti-CRY1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), anti-CRY2 (#HPA037577, Atlas Antibodies, Bromma, Sweden), and anti-β-actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA) antibodies.

Techniques: Expressing, Western Blot, Transfection, Infection, Mutagenesis

Journal: Scientific Reports

Article Title: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

doi: 10.1038/s41598-021-82287-0

Figure Lengend Snippet: CSAC obliterated molecular clock oscillation ex vivo. ( a ) Timeline of organotypic slice culture preparation and viral infection. ( b ) Representative confocal microscopy image of organotypically cultured SCN from mPER2::LUC;Cas9 mice. Scale bar: 100 μm. ( c – e ) Representative profiles of PER2::LUC mice with CSAC. Bioluminescence signals from SCN explant cultures infected with either sgLacZ ( c ), CSAC- Crys ( d ), or CSAC- Bmal1 ( e ) were continuously monitored. Red lines indicate forskolin treatment. ( f – h ) Oscillation amplitude ( f ), robustness ( g ), and period ( h ) in PER2::LUC slice cultures infected with AAV analyzed using Cosinor. * p < 0.05; ** p < 0.01 by Tukey’s post-hoc test ( n = 3–4 slices per group).

Article Snippet: The membranes were blocked in Tris buffered with Tween-20 (TTBS) with 1% bovine serum albumin (BSA) and incubated overnight at 4 °C in TTBS with 0.1% BSA with the following primary antibodies: anti-mPer1 (#AB2201, Merck Millipore), anti-PER2 (#AB2202, Merck Millipore), anti-BMAL1 (#NB100-2288, Novus, Centennial, CO, USA), anti-CRY1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), anti-CRY2 (#HPA037577, Atlas Antibodies, Bromma, Sweden), and anti-β-actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA) antibodies.

Techniques: Ex Vivo, Infection, Confocal Microscopy, Cell Culture

Journal: Scientific Reports

Article Title: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

doi: 10.1038/s41598-021-82287-0

Figure Lengend Snippet: SCN injection with CSAC. ( a ) Schematic representation of stereotaxic CSAC injection into the SCN. AAVs expressing sgRNAs targeting core clock components and the mCherry fluorescent marker were bilaterally injected into the SCN of Cas9-expressing mice. ( b ) Representative confocal microscopic image of the brain section containing CSAC-injected SCN. sgRNA-expressing cells are co-labeled with mCherry as a surrogate marker. Scale bar = 100 μm. ( c – e ) Immunohistochemical analysis of brain sections containing the SCN of mice injected with CSAC- Crys ( c ), CSAC- Pers ( d ), and CSAC- Bmal1 ( e ). Scale bar: 100 μm for ( b ) and 500 μm for ( c – e ). ( f – h ) Co-expression ratio of targeted clock proteins and CSAC. Percentage of clock protein-positive and mCherry-positive cells over mCherry-positive cells were quantified. ( f ) Co-expression percentages of CRY2 and mCherry are indicated for the sgLacZ or CSAC- Crys group. ( g ) Co-expression percentages of PER2 and mCherry are indicated for the sgLacZ or CSAC- Pers group. ( h ) Co-expression percentages of BMAL1 and mCherry are indicated for the sgLacZ or CSAC- Bmal1 group. Error bar = standard deviation (n = 1 mouse for sgLacZ; 3–4 mice for CSAC- Crys , CSAC- Pers , and CSAC- Bmal1 , respectively).

Article Snippet: The membranes were blocked in Tris buffered with Tween-20 (TTBS) with 1% bovine serum albumin (BSA) and incubated overnight at 4 °C in TTBS with 0.1% BSA with the following primary antibodies: anti-mPer1 (#AB2201, Merck Millipore), anti-PER2 (#AB2202, Merck Millipore), anti-BMAL1 (#NB100-2288, Novus, Centennial, CO, USA), anti-CRY1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), anti-CRY2 (#HPA037577, Atlas Antibodies, Bromma, Sweden), and anti-β-actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA) antibodies.

Techniques: Injection, Expressing, Marker, Labeling, Immunohistochemical staining, Standard Deviation

Journal: Scientific Reports

Article Title: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

doi: 10.1038/s41598-021-82287-0

Figure Lengend Snippet: Circadian locomotor activity in SCN-targeted mice. ( a – d ) Representative locomotor pattern displayed as a double plot of activity over 28 d in the control ( a ), CSAC- Crys - ( b ), CSAC- Pers - ( c ), and CSAC- Bmal1 - ( d ) injected mice. Gray indicates periods of constant darkness. ( e – h ) Chi-square periodogram of locomotor activity during constant darkness in control ( e ), CSAC- Crys - ( f ), CSAC- Pers - ( g ), and CSAC- Bmal1 - ( h ) injected mice. Gray indicates the periodogram of an individual mouse. The black line indicates the average spectrogram of each group. The red line indicates the significance threshold ( n = 4–8 mice per group).

Article Snippet: The membranes were blocked in Tris buffered with Tween-20 (TTBS) with 1% bovine serum albumin (BSA) and incubated overnight at 4 °C in TTBS with 0.1% BSA with the following primary antibodies: anti-mPer1 (#AB2201, Merck Millipore), anti-PER2 (#AB2202, Merck Millipore), anti-BMAL1 (#NB100-2288, Novus, Centennial, CO, USA), anti-CRY1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), anti-CRY2 (#HPA037577, Atlas Antibodies, Bromma, Sweden), and anti-β-actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA) antibodies.

Techniques: Activity Assay, Injection

Journal: Scientific Reports

Article Title: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

doi: 10.1038/s41598-021-82287-0

Figure Lengend Snippet: Circadian body temperature of SCN-targeted mice. ( a – d ) Representative body temperature profile shown as a double plot over 28 d for control ( a ), CSAC- Crys - ( b ), CSAC- Pers - ( c ), and CSAC- Bmal1 - ( d ) injected mice. Gray indicates periods of constant darkness. ( e – h ) Chi-square periodogram of body temperature during constant darkness in control ( e ), CSAC- Crys - ( f ), CSAC- Pers - ( g ), and CSAC- Bmal1 - ( h ) injected mice. Gray line indicates the periodogram of an individual mouse. Black line indicates the averaged spectrogram of each group. Red line indicates the significance threshold ( n = 4–8 mice per group).

Article Snippet: The membranes were blocked in Tris buffered with Tween-20 (TTBS) with 1% bovine serum albumin (BSA) and incubated overnight at 4 °C in TTBS with 0.1% BSA with the following primary antibodies: anti-mPer1 (#AB2201, Merck Millipore), anti-PER2 (#AB2202, Merck Millipore), anti-BMAL1 (#NB100-2288, Novus, Centennial, CO, USA), anti-CRY1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), anti-CRY2 (#HPA037577, Atlas Antibodies, Bromma, Sweden), and anti-β-actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA) antibodies.

Techniques: Injection

Journal: ACS Applied Materials & Interfaces

Article Title: Nanotopography Influences Host–Pathogen Quorum Sensing and Facilitates Selection of Bioactive Metabolites in Mesenchymal Stromal Cells and Pseudomonas aeruginosa Co-Cultures

doi: 10.1021/acsami.4c09291

Figure Lengend Snippet: Characterization of functional active coatings on Ti nanotopographies. (a) Representative SEM images of flat control, nanospike (NS), and nanonetwork (NN) together with surface height and surface area measurements (table); scale bar 2 μm. (b) Chemical structure of PEA. (c) Titanium, carbon, and oxygen spectra of PEA coated flat, NS, and NN taken by X-ray photoelectron spectroscopy (XPS) surfaces. Each color corresponds to the number on the PEA chemical structure. (d) 1 × 1 μm AFM micrographs from the flat, NS, and NN surfaces before coating (top row) and after coating with PEA for 90 s at 100 W using plasma polymerization, followed by fibronectin (FN) for 1 h (bottom row). (e) Roughness (Rq) and (f) contact angle (3 μL sessile water drop) measurements for flat, NS, and NN with PEA coating and PEA+FN coating. ELISA was used to quantify the amount of (g) FN and (h) BMP2 adsorbed onto flat, NS, and NN samples after one h of coating. (i) The percentage of BMP2 released into solution after PEA+FN coating, quantified at days 1, 3, 5, 7, 9, 12, and 14. (e–h) Average represented as bars with individual values and standard deviation. Comparison of differences was tested using a Kruskal–Wallis test with a p -value <0.05 (*) considered significant, and <0.001 (**) highly significant. Together these results show that the PEA+FN+BMP2 coating could be used on Ti flat, NS, and NN nanotopographies to test with bacteria, hMSCs, and co-cultures.

Article Snippet: FN and BMP2 levels in the supernatants were calculated using enzyme-linked immunosorbent assay (ELISA) duo-set Human Fibronectin (DY1918, R&D systems), and Human BMP2 (DY355, R&D systems), respectively, following instructions from the manufacturer.

Techniques: Functional Assay, Control, Spectroscopy, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison, Bacteria

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a Map of complement-mediated bactericidal activity. b The proportion of gut bacteria after 16s rRNA analyses of colon contents in normal CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice (pooled samples from six mice). Also see Fig. . c The proportion of gut bacteria after 16s rRNA analyses in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS. Also see Fig. . d The proportion of E. coli (Escherichia-Shigella) in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS ( n = 5). Also see Fig. S3C. e Flow cytometry of feces bacteria after staining using anti-LPS antibodies in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CDKO) mice after 2% DSS. f QPCR of colonic content (upper) and colonic tissues (lower) in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS ( n = 6). g FISH of E. coli in the gut tissues of CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS. One representative from six mice. Scale bar, 40 μM; Scale bar in bottom panel, 5 μM. Two side student’s t test in e and f ; * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Activity Assay, Bacteria, Flow Cytometry, Staining

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a E. coli clones in the normal human sera (N. Sera, Quidel), C3deficient sera (C3defi, Quidel), C1 deficient sera (C1 defi, Quidel) and inactive sera at different time points. *C3deficint sera vs normal sera. b E. coli clones in the normal sera (N. Sera, Quidel), CFD-deficient sera (CFD defi, Quidel) inactive sera at different time points. *CFD-deficient sera vs normal sera. c E. coli clones in the normal human sera after adding LPS (containing mannose), mannose or peptidoglycan. *LPS vs normal sera. d E. coli clones in the normal human sera after adding rReg4, rREG4 or mutated rREG4. *Reg4 vs normal sera. Only mrReg4 and only hrREG4, E.coli clones in inactive complement serum after adding mrReg4 or hrREG4. e E. coli clones in the normal human sera after adding different concentrations of rReg4. f E. coli clones in the normal human sera (N. sera) or CFD-deficient (CFD defi) sera after adding rReg4 (N. sera/rReg4 or CFDdefi/rReg4). *Normal sera/Reg4 vs normal sera. g E. coli clones in the normal human sera after adding rREG4, IgA or IgA+REG4. *IgA+Reg4 vs Reg4. Around 5 × 10 5 in a – c or 1 × 10 6 in d – g E. coli CFUs were added into 1 ml 10% sera. Bacterial suspension were diluted and plated on MacConkey agar plates. Plates were incubated overnight at 37 °C, and CFU were counted. Analysis of variance test used in a – g . * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. One representative of three independent experiments.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Clone Assay, Suspension, Incubation

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a Immunostaining of C3b, C5-9, Reg4, IgA, MASP1/3 and MASP2 on GFP-labeled E. coli in 2% DSS-treated GFP-labeled E. coli infused CFD fl/fl pvillin-cre T (CFDKO/0160) mice (Left) and statistic analyses (right, three images/slide, three slides/mouse, n = 6). Scale bar, 40 μM. No, number; Iso, isotypic antibod. b Flow cytometry of Reg4, IgA, MASP1/3 and MASP2 on the GFP-labeled E. coli in untreated GFP-labeled E. coli infused CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice and statistic analyses ( n = 3). c ELISA for detecting binding of Reg4 with LPS (left), Reg4 with IgA (middle) or IgA with LPS (right) in different concentration of Reg4 (left and middle) or IgA (right) coated plates d ELISA of C3 in different concentration of Reg4 (left) or IgA (right) with LPS coated plates. For complement resources, 5% normal human sera were added. e ELISA of C3b in the LPS, Reg4, IgA, LPS + Reg4 (LPS/Reg4), LPS + IgA (LPS/IgA), Reg4+IgA (Reg4/IgA) and LPS + Reg4+IgA (LPS/Reg4/IgA) coated plates. For complement resources, 5% normal human sera were added. Mann–Whitney U test in a ; Two side student’s t test in b ; ANOVA plus post-Bonferroni analysis in e ; Analysis of variance test in c and d . * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. In c – e , one representative of three independent experiments.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Immunostaining, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, MANN-WHITNEY

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 1. After M1 MΦs phagocytose NETs, P-SEP produced within M1 MΦs is released into extracellular space over time. (a) Cell morphology image of M1 MΦ phagocytosing PMA-NETs. (MG stain, magnification 1,000 ×). Scale bar: 10 µm. (b) Immunofluorescence staining results of M1 MΦ phagocytosing PMA-NETs. NETs indicated by arrows are citrullinated histone H3-positive. Scale bar: 10 µm. (c) Enlarged view of M1 MΦ phagocytosing PMA-NETs. Arrows indicate images of P-SEP production by M1 MΦs phagocytosing NETs. Scale bar: 5 µm. (d, e) After co-culturing M1 MΦs and NETs, cells were collected over time and analyzed for intracellular P-SEP expression using flow cytometry. P-SEP levels in culture medium supernatant were measured. (f) Western blotting of intracellular P-SEP expression intensity and P-SEP at 0, 15, and 180 min after NET phagocytosis by M1 MΦs. P-SEP protein levels were quantified using Image J. β-actin band intensity was used to correct P-SEP band intensity. The unprocessed western blot image is shown in Supplementary Fig. S11. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± standard deviation (SD; n = 3). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Produced, Staining, Immunofluorescence, Expressing, Flow Cytometry, Western Blot, Two Tailed Test, Standard Deviation

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 2. M1 MΦs phagocytose NETs and produce P-SEP. (a) P-SEP production after co-culture of M1 MΦs with PMA-NETs and DH5α-NETs was compared. (b) P-SEP production after co-culture of M1 MΦs or monocytes with PMA-NETs was compared. (c) Correlation between P-SEP production and NET ratio after M1 MΦ phagocytosed NETs. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 3). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Co-Culture Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 3. Evaluation of P-SEP levels in inhibiting NET formation, M1 MΦ phagocytosis, and protease inhibition. (a) Evaluation of culture medium supernatant P-SEP levels after adding various inhibitors. (b) Western blotting of P-SEP protein levels in M1 MΦs after adding various inhibitors. P-SEP protein levels were quantified using Image J. β-actin band intensity was used to correct P-SEP band intensity. The unprocessed western blot image is shown in Supplementary Fig. S12. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 3). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Inhibition, Western Blot, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 5. Identification of organs with MΦs producing P-SEP in a mouse model of sepsis. (a) Ly6c-positive cells were compared with positivity rate of Ly6C-positive M1 MΦs in each organ as M1 MΦs. M1 MΦs were the most abundant in the lungs of mice in the sham group, with a small number of M1 MΦs in the liver, spleen, and kidneys. Compared to the sham group, mice in the CLP group showed significantly increased Ly6C positivity in each organ, especially markedly in the lungs and liver. Each dot represents one mouse. (b) Comparing P-SEP expression in M1 MΦs in each organ between sham and CLP groups, intracellular P-SEP expression in each organ was lower in sham group mice, while P-SEP expression in M1 MΦs in lungs, liver, and spleen was significantly increased in CLP group mice. Each dot represents one mouse. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 5). **p < 0.01. P-SEP, presepsin; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Expressing, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 4. Evaluated blood NET ratio and blood P-SEP production in CLP-treated sepsis model mice. (a) Gross cecum findings for the sham group and CLP-treated sepsis model mice. As indicated by arrows, the ceca of CLP-treated mice were necrotic. (b, c) Comparison of viable bacteria calculated from colony counts. Each dot represents one mouse. (d, e) Blood NET ratio and P-SEP levels were evaluated in CLP-treated sepsis model mice. Each dot represents one mouse. All differences denoted by asterisks were subjected to two-tailed Student’s t-tests. Values are presented as mean ± SD (n = 5). **p < 0.01. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Comparison, Bacteria, Two Tailed Test

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 6. Images of P-SEP production in MΦ of lungs, liver, and kidneys. Immunofluorescence staining for P-SEP in MΦs in each organ showed that only a few MΦs in the sham group were positive for P-SEP, but P-SEP was markedly positive in MΦs from the lungs, liver, and kidney in the CLP group. Scale bar: 10 µm. P-SEP, presepsin; M1 MΦs, M1 macrophage; CLP, cecal ligation and puncture.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Immunofluorescence, Staining, Ligation

Journal: Scientific reports

Article Title: Identification of organs of origin of macrophages that produce presepsin via neutrophil extracellular trap phagocytosis.

doi: 10.1038/s41598-024-66916-y

Figure Lengend Snippet: Figure 7. Schematic diagram of the mechanism by which tissue MΦ produces P-SEP to phagocytose NETs. When bacteria infect blood vessels, neutrophils release NETs for biological defense. When MΦs in the lungs, liver, and spleen phagocytose increase NETs in the body, P-SEP is produced, and blood P-SEP levels are high. P-SEP, presepsin; NET, neutrophil extracellular trap; M1 MΦs, M1 macrophage.

Article Snippet: Details of reagents used in this study and their sources are as follows: Polymorphprep (Cat. No. 1114683; AxisShield, Dundee, Scotland), Phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, St. Louis, MO, USA), Escherichia coli DH5α competent cells (DH5α; Cat. No.9057; Takara Bio, Shiga, Japan), SYTOX Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechst 33,342 (Cat. No. 346-07,951; Fujifilm, Tokyo, Japan), EasySep Human Monocyte Isolation Kit (Cat. No. 19359; STEMCELL Technologies, Vancouver, BC, Canada), CellXVivo Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), DPI (Cat. No.81050; Cayman Chemical, Ann Arbor, MI, USA), Cytochalasin D (Cat. No.11330; Cayman Chemical), Pepstatin A (Cat. No. S7381; Selleck Biotech, Houston, TX, USA), May–Grünwald’s stain solution (Cat. No.15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No.15002; Mutokagaku), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No.15612; Mutokagaku), 4% paraformaldehyde phosphate buffer solution (Cat. No.09154-85; Nacalai Tesque, Kyoto, Japan), Skim milk powder (Cat. No. 190-12,865; Fujifilm Wako Pure Chemical, Tokyo, Japan), ChemiLuna One Ultra (Cat. No. 11644; Nacalai Tesque), Medetomidine hydrochloride (Domitol, ZENOAQ, Fukushima, Japan), Midazolam (Dormicum, Maruishi Pharmaceutical, Osaka, Japan), Butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Basel, Switzerland), and mouse presepsin ELISA Kit (PHC Holdings, Tokyo, Japan).

Techniques: Bacteria, Produced

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Flow cytometry quantification of live, CD45 + CD11b + immune cells recovered from WT and Ccl28 −/− mouse gut, blood, and bone marrow, before (Naive) and during STm infection (2 and 3 dpi). ( A ) Data indicate the relative abundance of neutrophils (CD11b + Ly6G + ) in the blood and bone marrow, as a proportion of total live CD45 + cells profiled. ( B ) Expression of CCR3 by eosinophils isolated from the gut and blood compartments of Naive and STm-infected (3 dpi) mice. The relative abundance of eosinophils ( C , CD11b + Ly6G − SiglecF + side scatter high ), macrophage-like F4/80 + CD11c − cells ( D , CD11b + Ly6G − SiglecF − F4/80 + CD11c − ), and conventional dendritic cell-like CD11c + F4/80 cells ( E , CD11b + Ly6G − SiglecF − CD11c + F4/80 − ), as a proportion of total live CD45 + cells profiled from each tissue. Each data point represents measurements from one mouse, with filled points from WT and empty points from Ccl28 −/− mice. Data are derived from the same set of pooled experiments presented in . Bars represent the median. Comparisons between WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data. ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Flow Cytometry, Infection, Expressing, Isolation, Derivative Assay, MANN-WHITNEY

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Surface expression of ( A, C ) CCR3 or ( B, D ) CCR10 on murine neutrophils obtained from ( A, B ) the gut, blood, and bone marrow (BM) 3 dpi with STm, or ( C, D ) the bronchoalveolar lavage (BAL), blood, and bone marrow 1 dpi with Ab, analyzed by flow cytometry. Left panels show representative histograms of ( A, C ) CCR3 or ( B, D ) CCR10 expression on the surface of neutrophils (gated on live, CD45 + CD11b + Ly6G + cells) from ( A, B ) the gut (blue), blood (red), and bone marrow (BM; black) or ( C, D ) BAL (blue), blood (red), and bone marrow (BM; black). Right panels show the percentage of ( A, C ) CCR3 + or ( B, D ) CCR10 + neutrophils obtained from ( A, B ) gut, blood, and BM or ( C, D ) BAL, blood, and BM. Data are from six independent experiments. ( E–H ) Uninfected bone marrow neutrophils were unstimulated or treated with the indicated stimuli for 4 hr. Surface expression of ( E, G ) CCR3 and ( F, H ) CCR10 on neutrophils was determined by flow cytometry. Left panels show representative histograms of ( E, G ) CCR3 or ( F, H ) CCR10 surface expression after stimulation with: ( E, F ) cytokines IFNγ + TNFɑ + GM-CSF (blue); fMLP (magenta); phorbol 12-myristate 13-acetate (PMA) (purple); lipopolysaccharide (LPS) (red); ( G, H ) cytokines IFNγ + TNFɑ + Granulocyte-macrophage colony stimulating factor (GM-CSF, blue); beads alone (magenta); cytokines plus beads (red). Right panels show the percentage of ( E, G ) CCR3 + or ( F, H ) CCR10 + neutrophils following stimulation with the indicated stimuli. US = unstimulated. Data shown are pooled from two independent experiments. ( I, J ) Bone marrow cells enriched for neutrophils were infected with opsonized STm at a multiplicity of infection (MOI) = 10 for 1 hr with (violet) or without (red) pretreatment with cytochalasin D for 30 min before infection. Surface expression of ( I ) CCR3 or ( J ) CCR10 was determined by flow cytometry. Data are from two independent experiments. Left panels show representative histograms of surface receptor staining on neutrophils, and right panels show the percentages. ( A–J , right panels) Bars represent the mean ± standard deviation (SD). ( A–D ) Data were analyzed by one-way analysis of variance (ANOVA) for paired samples (non-parametric Friedman test), assuming non-normal distribution and non-equal SD given the differences in the variance among the groups, followed by Dunn’s multiple comparisons test. ( E–J ) Data were analyzed by one-way ANOVA for paired samples, applying the Greenhouse–Geisser correction given the differences in variance among the groups; Bonferroni’s multiple comparison test was performed to compare between relevant stimulation conditions. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Expressing, Flow Cytometry, Infection, Staining, Standard Deviation, Comparison

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Surface expression of CCR3 and CCR10 on neutrophils obtained from the gut of WT mice ( n = 19, pooled from six independent experiments) infected with STm for 3 days, analyzed by flow cytometry. ( B ) Percentage of CCR3 + and CCR10 + neutrophils obtained from the gut, blood, and bone marrow of Ccl28 +/+ ( n = 19) and Ccl28 −/− mice ( n = 14) infected with STm for 3 days, analyzed by flow cytometry. ( C ) Surface expression of CCR3 and CCR10 on neutrophils obtained from the bronchoalveolar lavage (BAL) of WT mice ( n = 8, pooled from two independent experiments) infected with Ab for 1 day, analyzed by flow cytometry. ( D ) Percentage of CCR3 + neutrophils (WT n = 9; Ccl28 −/− n = 8) and CCR10 + neutrophils (WT n = 4; Ccl28 −/− n = 4) obtained from the BAL, lung, blood, and bone marrow of WT and Ccl28 −/− littermates infected with Ab for 1 day, analyzed by flow cytometry. ( A, C ) Left panels show representative contour plots, and right panels show the percentages of neutrophils expressing the indicated receptor on their surface. Symbols represent data from individual mice, bars represent the geometric means.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Expressing, Infection, Flow Cytometry

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Neutrophils enriched from wild-type mouse bone marrow were infected at multiplicity of infection (MOI) = 10 for 5 min to 4 hr with ( A, B ) opsonized Salmonella enterica serovar Typhimurium (STm) or ( C, D ) Acinetobacter baumannii (Ab). ( A, C ) Surface CCR3 or ( B, D ) intracellular CCR3 staining was detected by flow cytometry. Connected symbols represent data from neutrophils collected from the same mouse under different stimulation conditions. Neutrophils were obtained from ( E ) the gut, blood, and bone marrow 3 dpi with STm or ( F ) bronchoalveolar lavage (BAL), blood, and bone marrow 1 dpi with Ab. Surface (clear histograms) or intracellular (filled histograms) CCR3 expression was analyzed by flow cytometry. ( A–F ) Left panels show representative histograms, and right panels show the percentage of neutrophils expressing CCR3 on their surface (clear bars) or intracellularly (filled bars). Bars represent the mean. Data were analyzed by paired t test ( A–D ) or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test ( E, F ) on log-transformed data. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Infection, Staining, Flow Cytometry, Expressing, Comparison, Transformation Assay

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Murine bone marrow neutrophils were stimulated with IFNγ + TNFɑ + GM-CSF for 4 hr before adding 1 × 10 6 cells to the upper compartment of a transwell chamber for chemotaxis assays. Each of the chemokines (CCL28, CCL11, or CXCL1), or no chemokine (NC), was placed in separate lower compartments. The transwell plate was incubated for 2 hr at 37°C. Cells that migrated to the lower compartment were enumerated by flow cytometry. Neutrophil chemotaxis index was calculated by taking the number of cells that migrated in response to a chemokine and dividing it by the number of cells that migrated in the absence of a chemokine. Data are from four independent experiments. ( B, C ) Infection of bone marrow neutrophils. ( B ) Opsonized STm (1 × 10 7 CFU) or ( C ) opsonized Ab (1 × 10 7 CFU) were cultured alone, or added to bone marrow neutrophils (1 × 10 6 cells) stimulated with CCL28, CCL11, or no chemokine, for 2.5 hr (STm) or 4.5 hr (Ab) at 37°C. Neutrophils were lysed with 1% Triton-X and surviving bacteria were enumerated by plating serial dilutions. Percentage of bacterial survival was calculated for each condition by taking the CFU from bacteria incubated with neutrophils and dividing it by the CFU from bacteria incubated without neutrophils, multiplied by 100. Data shown for each infection comprise three independent experiments. Bars represent the mean ± standard deviation (SD). ( D ) The effect of the CCR3 antagonist SB328437 on neutrophil-mediated STm killing was evaluated by performing the experiment as described in panel ( B ), with or without the antagonist. Data shown comprise three independent experiments. ( E–G ) Reactive oxygen species (ROS) production (2′,7′-dichlorodihydrofluorescein diacetate [H 2 DCFDA] conversion to fluorescent DCF) detected by flow cytometry in bone marrow neutrophils infected with STm as described in panel ( B ). In ( F, G ), cells were stimulated with CCL28 in the presence of an anti-CCR3 antibody, an anti-CCR10 antibody, or isotype controls. Left panels show representative histograms, and right panels show the percentage of ROS + neutrophils in the indicated treatment groups. ( H, I ) Neutrophil extracellular trap (NET) formation detected by fluorescence microscopy using Helix dye in human neutrophils activated with platelets. Cells were unstimulated (no chemokine, NC), stimulated with CCL28 alone, or with CCL28 and the CCR3 agonist SB328737 and/or the CCR10 agonist BI-6901, as indicated. ( H ) Representative images of fluorescence microscopy with DAPI (blue) and Helix (green). ( I ) Quantification of NETs represented as percentage of cells forming NETs based on observed morphology. Connected circles represent NET abundance in cell populations from the same donor following different indicated treatments. ( A–E ) Bars represent the mean ± SD. ( A–C ) Data were analyzed by non-parametric analysis of variance (ANOVA) (Kruskal–Wallis’s test), assuming non-equal SD given the differences in the variance among the groups, followed by Dunn’s multiple comparisons test. ( D, I ) Data were analyzed by ratio paired t test. ( E–G ) Log-transformed data were analyzed by one-way ANOVA for paired samples. Greenhouse–Geisser correction was applied in F and G given the differences in variance among the groups. Tukey’s multiple comparison test was performed to compare all conditions to each other. ( I ) Ratio paired t tests were used to compare NET levels in samples from the same donor. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Chemotaxis Assay, Incubation, Flow Cytometry, Infection, Cell Culture, Bacteria, Standard Deviation, Fluorescence, Microscopy, Transformation Assay, Comparison

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: As indicated, cells were unstimulated (NC), stimulated with CCL28 alone, or with CCL28 and the CCR3 antagonist SB328437 and/or the CCR10 antagonist BI-6901 (as in ). ( A ) Representative contour plots, and ( B ) percentage of Helix + MPO + neutrophils in the indicated treatment groups. Connected circles represent NET abundance in cell populations from the same donor following different indicated treatments. Ratio paired t tests were used to compare NET levels in samples from the same donor. Significant changes are indicated by *p ≤ 0.05; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet:

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Sequencing, Generated, CRISPR, Isolation, Blocking Assay, In Vitro, Control, Recombinant, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, cDNA Synthesis, Incubation, Selection, Software, Infection, RNA Extraction, Staining, Immunofluorescence, Bacteria, Protease Inhibitor, Inhibition, Preserving, Cell Isolation, Extraction

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: MSCs were exposed to increasing LPS concentrations, thereafter washed and co‐cultured with PMA‐activated neutrophils. Incubation of neutrophils with PMA alone served as a positive control and DMSO‐treated neutrophils as a negative control. Scale bars: 50 μm. Quantification of NET‐bound elastase indicative of NET formation. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA‐histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS‐primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non‐primed MSC (middle panels) or PBS‐injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10 μm (upper row) and 50 μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Cell Culture, Incubation, Positive Control, Negative Control, Injection, Expressing, Activation Assay, Control, Comparison

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Graph shows quantification of ROS by measuring fluorescence intensity of DHR123 dye. The MSCs were primed with increasing concentrations of 10 ng/ml, 100 ng/ml, and 1,000 ng/ml LPS followed by co‐culture with neutrophils. The MSCs were then incubated with ROS‐specific dye DHR123, and the fluorescence was read after 60 min of incubation at 488/520 nm with spectrophotometer. Incubation of neutrophils with PMA alone served as a positive control and DMSO‐treated neutrophils as a negative control. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Fluorescence, Co-Culture Assay, Incubation, Spectrophotometry, Positive Control, Negative Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: A Hierarchical clustering analysis of RNAseq expression profile from MSCs stimulated with LPS for 6 and 24 h. The heat‐map shows the gene expression profile of non‐primed MSCs and MSCs which were LPS primed for 6 and 24 h. Red color represents up‐regulation, while blue color depicts down‐regulation in gene expression; each data point represents FPKM in log 2 value. B, C Pathway analysis depicts that cytokine–cytokine receptor interactions, genes encoding secreted soluble factors, an ensemble of genes encoding extracellular matrix, chemokine receptors which bind distinct chemokines, GPCR ligand binding, cytokine and inflammatory response, chemokine signaling pathway and IL‐5 signaling are the most dominant pathways as assessed by transcriptional changes when non‐primed and LPS‐primed MSCs were compared at 6 and 24 h after LPS priming. D Venn diagram analysis displays up‐regulated and down‐regulated gene numbers indicated in blue and pink colors, respectively. Purple color indicates the number of shared genes of the up‐regulated and down‐regulated genes at 6 h and 24 h after LPS stimulation. This implies rapid changes of expression of the same genes. E–G Validation of the selected genes uncovered from global RNAseq analyses by qRT–PCR analyses displays up‐regulation of CXCL‐6, IL‐8, and IL‐1β after LPS treatment. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. H, I ELISA results depict an up‐regulated expression of CXCL‐6 after 24 h of LPS treatment, while a significantly increased IL‐8 expression after 6 and 24 h of LPS treatment. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. J Western blot analysis showed up‐regulation of IL‐1β expression upon LPS priming of LPS‐primed MSCs. Actin served as control. Densitometry graph shows significant increase after 6 h of LPS stimulation compared to non‐primed MSCs. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Expressing, Gene Expression, Ligand Binding Assay, Biomarker Discovery, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Human MSCs were treated with LPS (100 ng/ml) for 24 h. The mRNA expression of MyD88 and IL‐6 was assessed by qRT–PCR analysis at 24 h. MyD88 (upper left panel) and IL‐6 gene expression (upper right panel) revealed an increase in MyD88 and IL‐6 in LPS‐primed MSCs. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. The TLR4 signaling pathway is depicted. Upon LPS binding, conformational changes in the TLR4 receptor led to recruitment of intracellular TLR domains harboring adaptor proteins. A bifurcation in MyD88‐dependent signaling converges to NF‐κB and pro‐inflammatory cytokine release, among them IL‐6, and the TRIF signaling pathway relaying its signals to downstream effectors like the transcription factor IRF which transactivates type 1 interferons. The right panel depicts representative Western blot analyses with a time‐dependent regulation of TLR4 and components of the TLR4 signaling pathway after exposure of MSCs with LPS. p65, a component of the heterodimeric NF‐κB transcription factor, and the IRF‐1 transcription factor are swiftly induced at 0.5 h, but down‐regulated to basal levels at 6 h after LPS challenge of MSCs. The target genes of NF‐κB are induced at 0.5 h, and this induction is differentially maintained. The expression of indicated proteins was analyzed in the cell lysates from LPS (100 ng/ml)‐ or PBS‐treated MSCs, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Binding Assay, Western Blot

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS‐primed MSCs into wounds increased the expression of CXCL6, IL‐8, and IL‐1β. Graphs display numbers of double‐positive cells for h‐β2M + CXCL6, h‐β2M + IL‐8, and h‐β2M + IL‐1β, respectively. MSC‐injected wounds served as controls. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50 μm. Results show that LPS‐primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL‐8 in mice and is known as neutrophil chemoattractant. Similar results were found for IL‐1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP‐2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double‐positive cells for Ly6G MIP/KC, IL‐1β, and LIX, respectively. PBS and non‐primed MSC‐injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Scale bars: 50 μm.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Injection, Expressing, Comparison, Functional Assay, Sequencing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: MSCs were either treated with a scrambled control siRNA (Scr. siRNA) or with four different siRNAs targeting the TLR4 receptor (siRNA smart pool). Forty‐eight hours after transfection, TLR4‐silenced and non‐silenced MSCs were analyzed for RNA expression by qPCR. A representative experiment is depicted. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. Seventy‐two hours after transfection, TLR4‐silenced and non‐silenced MSCs were treated with 100 ng/ml LPS for 16 h and subjected to Western blot analysis for protein expression. Densitometric assessment shows the percentage of the change in TLR4 expression in TLR4‐silenced and non‐silenced MSC. A representative experiment is depicted. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Control, Transfection, RNA Expression, Western Blot, Expressing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative microphotographs of TLR4‐silenced and non‐silenced MSCs primed with LPS and co‐cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA‐histone, red). Of note, TLR4‐silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non‐silenced, LPS‐primed MSCs co‐cultured with PMA‐activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co‐culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50 μm. Quantitative assessment of NET‐bound elastase employing a specific ELISA in identical experimental setting as described in Fig . Similarly, to immunostaining, a significant reduction of NET‐bound elastase was observed in TLR4‐silenced LPS‐primed MSCs when co‐cultured with activated neutrophils as opposed to high NET‐bound elastase in non‐silenced LPS‐primed MSCs co‐cultured with activated neutrophils. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates. High‐resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS‐primed MSC co‐cultured with PMA‐activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non‐primed MSCs co‐cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4‐silenced LPS‐primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4‐silenced and non‐primed MSCs display reduced NETs in co‐cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA‐activated neutrophils alone served as a positive control with highly enhanced NETs (upper row, outer right panel) and DMSO‐treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil‐derived granules. Scale bars: 0.1 μm

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Cell Culture, Immunostaining, Negative Control, Positive Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Activation Assay, Derivative Assay

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative clinical pictures of murine wounds at 0, 3, 5, 7, and 10 days after wounding. Enhanced wound healing in the LPS‐primed MSCs group as opposed to all other groups. Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non‐primed MSCs, LPS‐primed MSCs, non‐primed scrambled siRNA‐treated (Scr) MSCs, LPS‐primed Scr MSCs, and LPS‐primed TLR4‐silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one‐way ANOVA. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G + neutrophils (green) and F4/80 + macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Double‐stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bar: 100 μm. Quantification of Ly6G and F4/80 double‐positive cells on sections of differently injected day 1 wounds. Wounds were injected as described in (A). Double‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Quantification of Ly6G + neutrophils and F4/80 + macrophages on sections of differently injected day 1 wounds. Wounds were injected as described in (A). Single‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double‐stained for TGFβ‐1 (red) and for F4/80 + macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Scale bar: 50 μm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α‐SMA (indicative of myofibroblasts differentiation) after injection of LPS‐primed MSCs, non‐primed MSCs, LPS‐primed TLR4‐silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bars: 50 μm. Double‐positive macrophages stained for TGFβ‐1 and F4/80 were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Quantitative analysis of CD31‐positive endothelial cells in sections of wounds injected with PBS, non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced LPS. Cell counting was performed on immunostained wound sections; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depict enhanced α‐SMA protein expression in wounds injected with LPS‐primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Confocal Microscopy, Injection, Staining, Double Staining, Control, Comparison, Cell Counting, Western Blot, Expressing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative photomicrographs of sections from differently injected day 3 wounds stained for Ly6G + neutrophils (green) and caspase 3 (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 3 wounds injected with PBS, non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Double‐stained cells indicate apoptotic neutrophils. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bars: 50 μm. Quantification of Ly6G and caspase double‐positive cells on sections of differently injected day 3 wounds. A significant increase in double‐positive cells (Ly6G and caspase 3) in sections of wounds injected with LPS‐primed MSCs as opposed to wounds injected with LPS‐primed TLR4‐silenced MSCs and PBS control. Double‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Injection, Staining, Double Staining, Comparison, Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Graphical summary, depicting the molecules involved in sensing, signaling, and raising an adaptive response in MSCs. MSCs sense bacterial intruders as modeled by the key molecule LPS, a widely distributed PAMP and wall component of Gram‐negative bacteria. LPS is sensed via the TLR4 receptor, and the signal is relayed via MyD88 to downstream effectors which, in consequence, shape the adaptive MSC response. This response is enforced by fundamental transcriptomic reprogramming which is responsible for the release of factors critical for neutrophil and macrophage recruitment to the wound site. The adaptive response of LPS‐primed MSCs depicts a bifurcation with the activation of neutrophils with enhanced microbicidal NET formation and ROS release to directly counteract invading bacteria. In addition, in a second line of tissue protection and repair, apoptotic neutrophils are phagocytosed by macrophages. Neutrophil engulfment constitutes a strong signal for macrophages to release TGFβ‐1 which subsequently enhances differentiation of myofibroblasts and accelerates wound contraction and thus wound closure. Acceleration of wound closure together with enhanced NET formation and ROS release effectively counteracts microbial invasion. These data may stimulate new avenues to refine MSC‐based therapies for difficult‐to‐heal wounds and/or infected wounds.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Bacteria, Activation Assay, Infection

Journal: Molecular cell

Article Title: The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

doi: 10.1016/j.molcel.2020.06.014

Figure Lengend Snippet: (A) Schematic of samples prepared for mass spectrometry. dCas9-BirA* programmed with the on-target gRNA, Cas9-BirA* programmed with the on-target guide, and dCas9-BirA* programmed with a NT sgRNA were incubated with a 10-fold molar excess of plasmid and then added to HSS containing the ARS and biotin. Biotinylated proteins were isolated with Streptavidin-coupled beads and identified through mass spectrometry.

Article Snippet: Cas9, FACT, RNA, and Donor DNA Preparation Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal sequences and an HA tag at the C-terminus ( Lin et al., 2014 ) was expressed in Rosetta2 DE3 (QB3-Berkeley Macrolab) cells.

Techniques: Mass Spectrometry, Incubation, Plasmid Preparation, Isolation

Journal: Molecular cell

Article Title: The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

doi: 10.1016/j.molcel.2020.06.014

Figure Lengend Snippet: (A) Time course of Cas9 RNPs programmed against a linear DNA substrate in a 1:2 molar ratio in buffer, HSS with ATP, or HSS with CIP and ATPγS.

Article Snippet: Cas9, FACT, RNA, and Donor DNA Preparation Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal sequences and an HA tag at the C-terminus ( Lin et al., 2014 ) was expressed in Rosetta2 DE3 (QB3-Berkeley Macrolab) cells.

Techniques:

Journal: Molecular cell

Article Title: The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

doi: 10.1016/j.molcel.2020.06.014

Figure Lengend Snippet: (A) HDR rates from amplicon-NGS sequencing of VEGFA 0, 1, 2, 3, 6, 9, 12, and 24 hours after electroporation of Cas9 RNPs in the presence of an HDR donor (N = 3 biological replicates). Data are represented as mean ± standard deviation. See also Figure S3E.

Article Snippet: Cas9, FACT, RNA, and Donor DNA Preparation Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal sequences and an HA tag at the C-terminus ( Lin et al., 2014 ) was expressed in Rosetta2 DE3 (QB3-Berkeley Macrolab) cells.

Techniques: Amplification, Sequencing, Electroporation, Standard Deviation

Journal: Molecular cell

Article Title: The histone chaperone FACT induces Cas9 multi-turnover behavior and modifies genome manipulation in human cells

doi: 10.1016/j.molcel.2020.06.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cas9, FACT, RNA, and Donor DNA Preparation Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal sequences and an HA tag at the C-terminus ( Lin et al., 2014 ) was expressed in Rosetta2 DE3 (QB3-Berkeley Macrolab) cells.

Techniques: Purification, Recombinant, Amplification, Sequencing, Plasmid Preparation, Software

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: Overview of the high-content screening and analysis. (A) Summary of RNAi screening workflow. Reverse transfection of HeLa cells was performed in 384-well format for 72 h, followed by 48-h infection with GFP-expressing B. abortus , PFA fixation, and staining of HeLa cells with DAPI and DY-547−phalloidin before automated imaging. GW, genomewide. (B) Image analysis was performed with CellProfiler to segment nuclei and bacteria and to extract measurements. (C) Accurate association of segmented bacteria to nuclei enables quantitative single-cell measurements. The naive association (middle image) of segmented pathogen can be affected by oversplitting in dense cell populations (left image). Our proposed solution (right image) based on a nucleus attraction score. (D) The plate histogram shows the bimodal distribution of integrated GFP intensity corresponding to Brucella replication. Intensity on the x axes is log 2 scaled to account for exponential growth. The normal distribution fitted (red curve) to the kernel density estimation of the histogram allows us to compute a robust binary infection threshold (dashed line) separating HeLa cells with (right) and without (left) replicating Brucella . Associated are samples of single-cell images corresponding to the intervals of the intensity distribution (for more details, see Materials and Methods).

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: High Content Screening, Transfection, Infection, Expressing, Staining, Imaging, Bacteria, Quantitative Single Cell

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: Entry assay identifies new components required for postentry processes during Brucella infection. (A) Representative images from the entry assay showing nuclei (DAPI) of HeLa cells and intracellular Brucella abortus (GFP) for control condition (mock) and cells treated with siRNAs against CDC42 or VPS35. HeLa cells were infected with B. abortus expressing GFP under a tetracycline-inducible system for 8 h (see Materials and Methods). Bars = 100 μm. (B) Scatter plot in double logarithmic scale showing infection scores measured for the entry assay (8 hpi) versus endpoint assay (48 hpi), normalized to the respective mock data set ( Table S3 ). For the entry assay, cells containing single bacteria were considered infected, and the final readout is proportional to the median number of bacteria per infected cells. For the endpoint assay, only cells containing replicating bacteria were considered infected ( Fig. 1 and Materials and Methods). Each data point corresponds to the average of all siRNAs or esiRNAs targeted against the gene of interest ( n = 3). The straight fit (oblique line, r 2 = 0.763) indicates a globally high correlation between both assays. The blue region shows all points within 1 standard deviation (SD) of the fitted data. The genes falling out of this range are marked in red. For ease of visualization, only the averaged values over all RNAi products targeting a given gene are displayed.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: Infection, Control, Expressing, End Point Assay, Bacteria, Standard Deviation

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: The VPS retromer is a key component of Brucella intracellular trafficking. (A) Schematic representation of the retromer components and their prominent interactors. (B) Schematic representation of the shRNA constructs used in panel C. The gray box on the shVPS35/rescue construct indicates the silent mutations that prevent base-pairing with the coexpressed shRNA . (C) Infection index from transfected cells. Displayed are the averaged infection index and associated standard deviation after 48 h of Brucella infection. Data were normalized to the YFP-only condition ( n = 3). Values that are statistically significantly different from the value for the scrambled YPF-only condition as determined by paired t test are indicated by an asterisk (*, P value of ≤0.01; ns, not significant). (D) Dot box representation of the z-scored infection score for components of the retromer and interactors, including the positive-control ARPC3. Asterisks indicate statistically significant differences from the values for the scrambled siRNA-treated bacteria (control [Ctrl]) as determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple-comparison test (**, P value of ≤0.001; ns, not significant). (E) Normalized CFU recovered from siRNA-treated cells at 6, 20, or 44 hpi. The presented data correspond to CFU count normalized to control, siRNA-treated cells ( n = 3). Significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test (*, P ≤ 0.01; **, P ≤ 0.001; ns, not significant). (F) Western blot analysis of the indicated proteins in total lysate of HeLa cells treated with siRNA targeting the designated genes, 72 h posttransfection. Displayed is a representative example of an experiment performed in biological triplicate ( n = 3). See Table S4 for the matching averaged intensity quantification.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: shRNA, Construct, Infection, Transfection, Standard Deviation, Positive Control, Bacteria, Control, Comparison, Western Blot

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: VPS35 is required for Brucella to escape the lysosomal pathway. (A) Immunofluorescence approach used to quantify localization of Brucella within LAMP-1-positive vesicles, illustrated with a representative example of control-treated cells 6 hpi. Individual channels and merged picture are presented. Arrows indicate examples of colocalization of bacteria with LAMP-1-positive compartments. Staining of Brucella LPS (anti-LPS [αLPS]) was used to confirm the presence of LAMP-1 in direct proximity of the bacterial surface. Asterisks indicate examples of LAMP-1-negative Brucella . (B) Representative images from Brucella infected cells either mock transfected (ctrl) or after VPS35 knockdown (siVPS35). Samples were fixed 6 and 18 hpi. For clarity, only the LAMP-1 (cyan) and dsRed (magenta) channels are presented. Bars, 5 μm. (C) Global quantification of LAMP-1-negative Brucella. Displayed are the average and associated standard deviation for more than 500 bacteria and more than 50 HeLa cells per time point and condition ( n = 3). (D) Single-cell data representation of the data presented in panel C. Displayed is the distribution of LAMP-1-positive Brucella per cell as a function of the total number of bacteria counted in that given cell.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: Immunofluorescence, Control, Bacteria, Staining, Infection, Transfection, Knockdown, Standard Deviation