Journal: Alzheimer's & Dementia

Article Title: Gut‐derived bacterial vesicles carrying lipopolysaccharide promote microglia‐mediated synaptic pruning

doi: 10.1002/alz.70331

Figure Lengend Snippet: bEVs induced C1q‐mediated synaptic pruning through activating Piezo1. (A and B) Western blot and quantitative analysis demonstrate the protein levels of C1q in HMC3 cells treated with PBS or bEVs ( N = 3 independent rounds of experimentation. In each round, three wells of HMC3 cells per group were measured and averaged to generate a single data point). (C) qPCR results and quantitative analysis demonstrate mRNA levels of C1q and C3 in HMC3 cells treated with PBS or bEVs ( N = 5 independent rounds of experimentation. In each round, four wells of HMC3 cells per group were measured and averaged to generate a single data point). (D and E) Western blot and quantitative analysis demonstrate protein levels of C1q in the cortex and hippocampus of 2‐month‐old mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 6 and 8 mice for PBS and bEVs groups respectively). (F and G) qPCR results and quantitative analysis demonstrate mRNA levels of C1q and C3 in the cortex and hippocampus of 2‐month‐old mice treated with or without bEVs via tail vein injection for 24 h ( N = 3 and 5 mice for PBS and bEVs group, respectively). (H and I) Western blot and quantitative analysis demonstrate protein level of C1q in the hippocampus of 2‐month‐old mice treated with PBS, Pxb, bEVs, or bEVs+PxB via brain stereotactic injection for 24 h ( N = 3 mice in each group). (J and K) Representative immunofluorescence images and quantitative analysis show the presence of synaptic puncta (PSD95+) in a primary neuron and microglia cocultured system, and coculture cells were pretreated with ANX005 for 1 h, followed by treatment with PBS or bEVs ( N = 3 independent rounds of experimentation, each using primary neurons and microglia isolated from five different neonatal mice. In each round, six randomly captured neurons per well were measured and averaged to generate a single data point). (L and M) Western blot and quantitative analysis demonstrate protein levels of C1q in HMC3 cells pretreated with or without 5 µM GsMTx4 for 1 h, followed by bEV treatment ( N = 3 round of experimentation. In each round, four wells of HMC3 cells per group were measured and averaged to generate a single data point). Scale bar: 10 µm for J. Values are means ± SEM. ns, not significant; * p < .05. bEV, bacterial extracellular vesicle; PxB, polymyxin B.

Article Snippet: Antibodies used in Western blot included LPS (ab35654, Abcam, USA, 1:1000), OmpA (111120, Antibody Research, USA, 1:1000), CD9 (C‐4) (sc‐13118, Santacruz, USA, 1:1000), C1q (nbp1‐51139, Novus, USA; 11602‐1‐AP, Proteintech, China, 1:1000), C3 (21337‐1‐AP, Proteintech, China, 1:1000), CX3CR1 (nbp1‐76949, Novus, USA, 1:1000), SIRPα (14482‐1‐AP, Proteintech, China, 1:1000), CD47 (66301‐1‐AP, Proteintech, China, 1:1000), Piezo1 (15939‐1‐AP, Proteintech, China, 1:1000), anti‐GAPDH (G9545, Sigma Aldrich, USA, 1:5000), and anti‐β‐actin (66009‐1‐Ig, Proteintech, China, 1:10000).

Techniques: Western Blot, Injection, Immunofluorescence, Isolation

Journal: Alzheimer's & Dementia

Article Title: Gut‐derived bacterial vesicles carrying lipopolysaccharide promote microglia‐mediated synaptic pruning

doi: 10.1002/alz.70331

Figure Lengend Snippet: Myeloid‐specific Piezo1 deficiency mice resisted the synaptic loss and activation of complement system induced by bEVs. (A and B) Representative immunofluorescence images and quantitative analysis show Iba1 in the cortex and hippocampus of Piezo1 fl/fl and Piezo1 ∆LysM mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 6 mice in each group; five randomly taken images per mouse were measured and averaged into a single value). (C and D) Representative immunofluorescence images and quantitative analysis show the colocalization of SYN and PSD95 in the hippocampus of Piezo1 fl/fl and Piezo1 ∆LysM mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 6 mice in each group; three randomly taken images per mouse were measured and averaged into a single value). (E and F) Representative high‐magnification immunofluorescence images and quantitative analysis show the colocalization of SYN and PSD95 in the hippocampus of Piezo1 fl/fl and Piezo1 ∆LysM mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 6 mice in each group; five randomly taken images per mouse were measured and averaged into a single value). (G and H) Three‐dimensional reconstruction and surface rendering demonstrate larger volumes of PSD95 puncta inside Iba1‐positive microglia in the cortex and hippocampus of Piezo1 fl/fl and Piezo1 ∆LysM mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 6 mice in each group; six randomly captured microglia per mouse were measured and averaged into a single value). (I and J) Western blot and quantitative analysis demonstrate protein levels of C1q in the cortex and hippocampus of Piezo1 fl/fl and Piezo1 ∆LysM mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 3 Piezo1 fl/fl mice and N = 4 Piezo1 ∆LysM mice in both PBS and bEV group, respectively). (K and L) qPCR and quantitative analysis demonstrate mRNA levels of C1q and C3 in the cortex and hippocampus of Piezo1 fl/fl and Piezo1 ∆LysM mice treated with PBS or bEVs via tail vein injection for 24 h ( N = 3 Piezo1 fl/fl mice and N = 4 Piezo1 ∆LysM mice in both PBS and bEVs group, respectively). Scale bar: 25 µm for A, C, and G, 8 µm for E. Values are means ± SEM. * p < .05. bEV, bacterial extracellular vesicle; Cx, cortex; Hp, hippocampus; SYN, synaptophysin.

Article Snippet: Antibodies used in Western blot included LPS (ab35654, Abcam, USA, 1:1000), OmpA (111120, Antibody Research, USA, 1:1000), CD9 (C‐4) (sc‐13118, Santacruz, USA, 1:1000), C1q (nbp1‐51139, Novus, USA; 11602‐1‐AP, Proteintech, China, 1:1000), C3 (21337‐1‐AP, Proteintech, China, 1:1000), CX3CR1 (nbp1‐76949, Novus, USA, 1:1000), SIRPα (14482‐1‐AP, Proteintech, China, 1:1000), CD47 (66301‐1‐AP, Proteintech, China, 1:1000), Piezo1 (15939‐1‐AP, Proteintech, China, 1:1000), anti‐GAPDH (G9545, Sigma Aldrich, USA, 1:5000), and anti‐β‐actin (66009‐1‐Ig, Proteintech, China, 1:10000).

Techniques: Activation Assay, Immunofluorescence, Injection, Western Blot

Journal: Glycobiology

Article Title: Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types.

doi: 10.1093/glycob/cwl057

Figure Lengend Snippet: Fig. 1. Antigen-binding activity of antihuman CD20 IgG1s measured by ELISA. Human CD20þ B lymphoma cell line Raji microsomes were coated and incubated with various concentrations of antibody. Goat antihuman IgG1 polyclonal antibodies were used to detect CD20 binding. The results shown are for the antibodies produced by Lec13 A4 with L-fucose (open squares), Ms704 1A7-15 (closed squares), Lec13 A4 with L-fucose and swainsonine (open diamonds), Ms704 1A7-15 with swainsonine (closed diamonds), Ms704 1A7-15 with kifunensine (open triangles), and Lec1 R7 (open circles). All produced antibodies showed the same antigen-binding affinity as rituximab (closed circles).

Article Snippet: Cells were then washed and incubated with fluorescein isothiocyanate-conjugated polyclonal antibodies against human C1q (Acris Antibodies GmbH, Hiddenhausen, Germany) for 30 min. Stained cells were analyzed by flow cytometry using FACSCalibur.

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, Produced

Journal: Glycobiology

Article Title: Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types.

doi: 10.1093/glycob/cwl057

Figure Lengend Snippet: Fig. 3. FcgRIIIa-binding activity of anti-human CD20 IgG1s measured by ELISA. Goat antihuman IgG1 polyclonal antibodies were coated on 96-well immunoplates and incubated with equal amounts (0.5 mg/well) of sample antibodies and soluble human FcgRIIIa-His fusion protein [either (A) the FcgRIIIa-158Val allotype or (B) the FcgRIIIa-158Phe allotype]. The binding was detected by anti-Tetra.His antibody as the absorbance at 450 nm. Antihuman CD20s of the Fu(þ) complex type, the Fu(2) complex type, the Fu(þ) hybrid type, the Fu(2) hybrid type, and the high-mannose types M5 and M8,9 were employed as samples.

Article Snippet: Cells were then washed and incubated with fluorescein isothiocyanate-conjugated polyclonal antibodies against human C1q (Acris Antibodies GmbH, Hiddenhausen, Germany) for 30 min. Stained cells were analyzed by flow cytometry using FACSCalibur.

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Glycobiology

Article Title: Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types.

doi: 10.1093/glycob/cwl057

Figure Lengend Snippet: Fig. 8. C1q-binding activity of antihuman CD20 IgG1s. The C1q binding on human CD20þ B lymphoma cell line WIL2-S cells induced by the corresponding antihuman CD20s was measured by flow cytometry. The binding ability was represented as the median fluorescence intensity. Antihuman CD20s of the Fu(þ) complex type (open squares), the Fu(2) complex type (closed squares), the Fu(þ) hybrid type (open diamonds), the Fu(2) hybrid type (closed diamonds), and the high-mannose types M5 (open circles) and M8,9 (open triangles) were employed as samples.

Article Snippet: Cells were then washed and incubated with fluorescein isothiocyanate-conjugated polyclonal antibodies against human C1q (Acris Antibodies GmbH, Hiddenhausen, Germany) for 30 min. Stained cells were analyzed by flow cytometry using FACSCalibur.

Techniques: Binding Assay, Activity Assay, Cytometry

Journal: Diabetology & Metabolic Syndrome

Article Title: Single-cell RNA sequencing reveals roles of unique retinal microglia types in early diabetic retinopathy

doi: 10.1186/s13098-024-01282-3

Figure Lengend Snippet: Inflammatory and activation characteristics of microglial subtypes. A Three-dimensional UMAP plot of microglia and macrophages. The different colors correspond to different cell types. B Boxplot showing the marker genes of microglia and macrophages. According to their different gene expression profiles, the microglia were categorized into three groups: Egr2 + M1 microglia ( C1qa + , Ccr5 + , Egr2 + ), Egr2 − M1 microglia ( C1qa + , Ccr5 + , Egr2 − ) and M2 microglia ( C1qa + , Ccr5 − , Egr2 − ). Macrophages highly expressed Cxcr4 . C Immunofluorescence labeling for C1qa (red) and Ccr5 (green) and DAPI nuclear staining (blue) in the rat retina. M1 microglia are indicated by arrows, and M2 microglia are indicated by arrowheads (above, 63 ×). Immunofluorescence labeling for Ccr5 (red) and Egr2 (green) and DAPI nuclear staining (blue) in the rat retina. Egr2 + M1 microglia are indicated by arrows, and Egr2 − M1 microglia are indicated by arrowheads (below, 63 ×). Scale bar 20 µm. D Immunofluorescence labeling for C1qa (red) and Ccr5 (green) and DAPI nuclear staining (blue). M2 microglia are indicated by arrows (left, 20 ×). Immunofluorescence labeling for Ccr5 (red) and Egr2 (green) and DAPI nuclear staining (blue). Egr2 + M1 microglia are indicated by arrowheads, and Egr2 − M1 microglia are indicated by asterisks (right, 20 ×). Scale bar 50 µm. E Plot showing the pathways enriched in M2 microglia in GSEA. The threshold limits were a p value < 0.1 and a FDR q-value < 0.25. F Heatmap showing the average expression of inflammatory mediators in microglia and macrophages. The plot is ordered by the three gene categories (labels on left). High expression is in red, and low expression is in blue. G Bubble plots showing the expression of inflammatory cytokines in microglia subpopulations from different data sets. The size of each circle is proportional to the percentage of gene expression. High expression is shown in blue, and low expression is shown in gray. Mouse_2, Mouse_3 and Mouse_brain were obtained from normal mouse samples. H Bubble plot showing the expression of inflammatory mediators in Egr2 + M1 microglia and Egr2 − M1 microglia in different stages of DR. The size of each circle is proportional to the percentage of gene expression. High expression is shown in red/yellow, and low expression is shown in gray

Article Snippet: Antibodies used in this study were as follows: Purified mouse Complement Component C1qA Antibody (NOVUS NBP1-51,139); Rabbit Monoclonal EGR2 Antibody (JG78-39); Rat monoclonal [HEK/1/85a] to CCR5 (Abcam ab11464); Cy3–conjugated Affinipure Goat Anti-Mouse IgG(H + L) (SA00009-1); Goat Anti-Rabbit IgG(H + L), Mouse/Human ads-APC (SBA-4050-11S).

Techniques: Activation Assay, Marker, Gene Expression, Immunofluorescence, Labeling, Staining, Expressing

Journal: Diabetology & Metabolic Syndrome

Article Title: Single-cell RNA sequencing reveals roles of unique retinal microglia types in early diabetic retinopathy

doi: 10.1186/s13098-024-01282-3

Figure Lengend Snippet: Activation mechanism of microglia. A Volcano plot showing the DEGs between Egr2 + M1 microglia and Egr2 − M1 microglia. The adjusted P value and gene expression (log fold change) were used for the plot. B PPI network showing the highly expressed proteins of Egr2 + M1 microglia. The size and color represent the combined score of each protein. Significant proteins are marked with red circles. C Heatmap showing the enrichment of pathways in different subtypes of microglia in GSVA. High expression is in red, and low expression is in blue. D Violin plot showing the expression of inflammatory receptors in the three groups of microglia. E Gating strategy for sorting Egr2 + M1 cells. Egr2 + M1 cells are gated as Ccr5 + C1qa + Egr2 + cells. F The bar graph shows the qPCR results for Egr2 + M1 cells. * denotes a p-value of < 0.05, ** denotes a p-value of < 0.01 and *** denotes a p-value of < 0.001. The levels of inflammatory cytokines like Tnf and members of the AP-1 family were significantly elevated in Egr2 + M1 cells compared to normal cells

Article Snippet: Antibodies used in this study were as follows: Purified mouse Complement Component C1qA Antibody (NOVUS NBP1-51,139); Rabbit Monoclonal EGR2 Antibody (JG78-39); Rat monoclonal [HEK/1/85a] to CCR5 (Abcam ab11464); Cy3–conjugated Affinipure Goat Anti-Mouse IgG(H + L) (SA00009-1); Goat Anti-Rabbit IgG(H + L), Mouse/Human ads-APC (SBA-4050-11S).

Techniques: Activation Assay, Gene Expression, Expressing