Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: sWTA probe generation and validation. A) schematic representation of sWTA probe generation for all three glycoforms, i.e. RboP +β-1,4-GlcNAc, +α-1,4-GlcNAc, or + β-1,3-GlcNAc. All sWTA probes were made for detection in two different fluorescence channels using streptavidin conjugated to AF647 or BB515. B) dual sWTA probe labeling of protein A-coated beads coated with anti-β-GlcNAc RboP (clone 4497), anti-α-GlcNAc RboP (clone 4461), and isotype IgG1. Data in dot plots represent geometric mean fluorescence intensity (gMFI) signals (fluorophores: AF647 and BB515) on the beads. Q2 and Q4 comprise, respectively, double positive (dual sWTA probe labeling) and double negative (no sWTA probe binding) beads. Signals within Q1 and Q3 represent aspecific binding of, respectively, AF647 and BB515 streptavidin to beads. Histograms are included on the sides of the dot plots to visualize relative amounts of different bead populations within a fluorescent channel.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Biomarker Discovery, Fluorescence, Labeling, Binding Assay

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: sWTA specificity screening of pilot-scale produced mAbs. Binding profiles of 15 B cell-derived mAbs, expressed by HEK293T cells (production levels in ) as human IgG1, to streptavidin-coated beads immobilized with biotinylated sWTA to determine clone reactivity as measured by flow cytometry. Fluorescent signals are depicted in this figure as gMFI fold changes (mean + s.d. of three independent experiments) relative to the condition without antibodies to compensate for technical variation. One-way ANOVA was performed to determine significant binding to glycan-coated beads compared to empty beads. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Produced, Binding Assay, Derivative Assay, Flow Cytometry, Glycoproteomics

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Specificity verification of sWTA-reactive mAbs at equimolar level. Clones that displayed sWTA reactivity in the pilot screening (main ) were selected for large-scale production in HEK293 freestyle cells and purified through protein G agarose. Selected clones were categorized based on their α-GlcNAc A) or β-GlcNAc B) reactivity and relative binding capacities to sWTA beads were determined at a concentration of 3 μg/ml. Beads coated with polyrhamnose (PR) + β-1,3-GlcNAc and empty beads were used as controls for cross-reactivity and background, respectively. IgG1 binding to sWTA beads was measured by flow cytometry and data represent the gMFI mean + s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine significant binding of sWTA-reactive clones to glycan-coated beads compared to empty beads. ns not significant ** P < 0.01, **** P < 0.0001. Index sort data of all sorted clones can be found in .

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Clone Assay, Purification, Binding Assay, Concentration Assay, Flow Cytometry, Glycoproteomics

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Binding of sWTA-reactive mAbs to WTA on S. aureus surface. For each glycoform specificity, one mAb clone was selected to assess bacterial opsonization. A-D) binding of W1C11 (anti-α-GlcNAc), W1F10 (anti-β-GlcNAc, with preference for β-1,3-GlcNAc), W1G7 (anti-β-GlcNAc), and B12 (isotype control) to S. aureus strains N315 Δ spa A), N315 Δ spa Δ tarSP B), Newman Δ spa Δ sbi C), and Streptococcus pyogenes strain 5448 Δ gacH D). IgG1 binding to bacteria was measured using flow cytometry and data represent normalized mean gMFI + s.d. (isotype signals set to 1) of three independent experiments. N315 Δ spa Δ tarSP and S. pyogenes 5448 Δ gacH were included as controls for WTA GlcNAc (species) specificity.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Binding Assay, Control, Bacteria, Flow Cytometry

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Effector functions of anti-WTA clones towards S. aureus . A-C) C3b deposition by sWTA-reactive mAbs onto S. aureus strains N315 Δ spa A), N315 Δ spa Δ tarSP B), and Newman Δ spa Δ sbi C). Data represent C3b binding (normalized gMFI + s.d.) of three independent experiments as measured by flow cytometry. Fluorescent signals are depicted as a fold change relative to the condition without antibodies to compensate for variation in background signals between biological replicates. D) neutrophil-mediated phagocytosis of GFP-expressing S. aureus Newman Δspa Δsbi by anti-WTA mAbs. Displayed data represent percentages of GFP-positive neutrophils and are representative of three biological replicates (individual replicates can be found in ). Curves were generated using nonlinear dose–response fitting model. E) relative phagocytic capacities of the anti-WTA mAbs. Absolute IC 50 values were determined for each replicate individually using nonlinear dose–response fitting model. Black lines represent means of the IC 50 values which are depicted as data points. Assays (A-D) were performed in the presence of 1% IgG-/IgM-depleted human serum as complement source. Statistical differences compared to isotype were determined by one-way ANOVA. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Clone Assay, Binding Assay, Flow Cytometry, Expressing, Generated

Journal: Glycobiology

Article Title: Editor’s Choice Platform for identifying human glycan-specific antibodies against bacterial pathogens using synthetic glycan fragments

doi: 10.1093/glycob/cwaf064

Figure Lengend Snippet: Discovery and characterization of GAC-specific mAbs. A) schematic representation of sGAC probe generation for two glycoforms, i.e. PR and PR + β-1,3-GlcNAc. All sGAC probes were made for detection in two different fluorescence channels using streptavidin conjugated to Pe-Cy7 or BV421. B) sGAC probe binding to protein beads immobilized with goat polyclonal anti-GAC GlcNAc (Ab9191). N.B. no bead coat option was available to test PR specificity. Data in dot plots represent fluorescence signals (fluorophores: PE-Cy7 and BV421) on the beads. Q2 and Q4 comprise, respectively, double positive (dual sGAC probe labeling) and double negative (no sGAC probe binding) beads. Signals within Q1 and Q3 represent aspecific binding of, respectively, Pe-Cy7 and BV421 streptavidin to beads. C) specificity verification of sGAC-reactive mAbs at equimolar level. Clones were produced in HEK293 freestyle cells and purified through protein G agarose. Relative binding capacities to sGAC beads were determined at a concentration of 3 μg/mL. Beads coated with RboP +β-1,3-GlcNAc and empty beads were used as controls for cross-reactivity and background, respectively. IgG1 binding to sGAC beads was measured by flow cytometry and data represent the mean gMFI ± s.d. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine significant binding of sGAC-reactive clones to glycan-coated beads compared to empty beads. **** P < 0.0001. Index sort data of all sorted clones can be found in . D) binding of sGAC-reactive mAbs to natural GAC on Streptococcus pyogenes . For each glycoform specificity, one mAb clone was selected that showed evident binding to sGAC beads (panel C) and tested for bacterial opsonization. Titration of G1E8 (anti-PR), G1C4 (anti-β-1,3-GlcNAc PR), and B12 (isotype control) to S. pyogenes 5448 Δ emm1 . Bacterial opsonization was determined by measuring IgG1 binding to bacteria using flow cytometry and data represent normalized mean gMFI + s.d. (isotype signals set to 1) of three independent experiments. E) effector functions of anti-GAC clones towards S. pyogenes. C3b deposition by sGAC-reactive mAbs onto S. pyogenes 5448 Δ emm1 . Data represent C3b binding (normalized gMFI + s.d.) of three independent experiments and was measured by flow cytometry. Fluorescent signals are depicted as a fold change relative to the condition without antibodies to compensate for variation in background signals between biological replicates.

Article Snippet: Subsequently, beads were incubated with 1:1000 diluted AlexaFluor488-conjugated protein G ( P11065 , Thermo Scientific) or 1:500 PE-conjugated mouse anti-Human IgG1 Fc (HP6001; Southern Biotech).

Techniques: Fluorescence, Binding Assay, Labeling, Clone Assay, Produced, Purification, Concentration Assay, Flow Cytometry, Glycoproteomics, Titration, Control, Bacteria

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation