Journal: Brain Pathology

Article Title: Reduction in the neuronal surface of post and presynaptic GABA B receptors in the hippocampus in a mouse model of Alzheimer's disease

doi: 10.1111/bpa.12802

Figure Lengend Snippet: Regional and cellular distribution of GABA B1 in wild‐type and APP/PS1 mice . A‐H . Immunoreactivity for GABA B1 in the hippocampus of wild‐type and APP/PS1 mice at 12 months of age using a pre‐embedding immunoperoxidase method at the light microscopic level. In the CA1 and CA3 regions and dentate gyrus (DG), GABA B1 immunoreactivity was very similar both in the wild‐type and the APP/PS1 mice, regardless of accumulation of amyloid plaques (asterisks). Labeling for GABA B1 showed the highest intensity in the stratum lacunosum‐moleculare (slm) and molecular layer (ml) and weaker in the strata oriens (so) and radiatum (sr). Immunoreactivity for GABA B1 was also detected in interneurons throughout all layers (white arrows), with similar distribution pattern and labeling intensity in wild‐type and APP/PS1 mice. I,J . Immunoreactivity for β‐amyloid in wild‐type and APP/PS1 mice at 12 months of age, showing high accumulation of Aβ throughout all layers of the hippocampus. Abbreviations : CA1 region of the hippocampus; CA3, CA3 region of the hippocampus; DG, dentate gyrus; so, stratum oriens ; sp, stratum pyramidale ; sr, stratum radiatum ; slm, stratum lacunosum‐moleculare ; ml, molecular layer; gc, granule cell layer; h, hilus. Scale bars: A,B,I,J 200 µm; C‐H , 100 µm.

Article Snippet: An affinity‐purified polyclonal antibody against β‐amyloid (ref #2454, detecting human Aβ‐40 and Aβ‐42 peptides) raised in rabbit was obtained from Cell Signalling Technology (Leiden, The Netherlands).

Techniques: Labeling

Journal: iScience

Article Title: Once-monthly hemin suppresses inflammatory and autoreactive CD4 + T cell responses to robustly ameliorate experimental rheumatoid arthritis

doi: 10.1016/j.isci.2021.103101

Figure Lengend Snippet:

Article Snippet: Bovine type Ⅱ collagen , Chondrex , 20022.

Techniques: Recombinant, RNA Extraction, SYBR Green Assay, Software

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunostaining for HMGB1 (yellow) and bisbenzimide H 33258 (Hoescht, blue) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. (n=20) b, HMGB1 concentration in colonic mucus from HMGB1 WT and HMGB1 ΔIEC mice measured by ELISA. (n=6) c, Immunoblotting for HMGB1 in colonic mucus from HMGB1 WT and HMGB1 ΔIEC mice. (n=7) d, Immunostaining for HMGB1 (yellow) and Hoescht (blue) in Carnoy’s fixed proximal colon sections from SPF and GF C57BL/6 mice. Arrows indicate the epithelial surface. (n=6) e, Immunoblotting for HMGB1 in mucosal scrapings from SPF and GF C57BL/6 mice. (n=4) f, HMGB1 concentration in stool from SPF and GF C57BL/6 mice measured by ELISA. (n=6) Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one individual mouse. Scale bars, 100 µm. Original magnification 400x

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: Immunostaining of Carnoy’s fixed proximal colon sections from HMGB1 WT mice either using antibody treated with buffer (No peptide) or antibody incubated with the immunizing peptide (Peptide) to block HMGB1-specific staining. Scale bars, 100 µm. Original magnification 400x.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Incubation, Blocking Assay, Staining

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Fluorescence in situ hybridization (FISH) using the EUB338 probe (purple) and Hoescht (blue) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. Dotted lines are placed at the epithelial surface and inner edge of the microbial community. Straight line indicates distance between host tissues and microbial community. (n=12) b, Distance measured between epithelium and bacterial cells in images represented in (a). Each datapoint is an average of 5 measurements for one individual mouse. c, Quantitative PCR for the bacterial 16S rRNA gene in 1 cm of colonic tissue from HMGB1 WT and HMGB1 ΔIEC mice. (n=6) d, Invasion of green fluorescent protein (GFP) labeled E. coli (SWW33) into mucus isolated from HMGB1 WT or HMGB1 ΔIEC mice. Original magnification 200x (n=3; 3 replicates) e, Percentage of the total GFP signal in mucus from HMGB1 WT vs. HMGB1 ΔIEC mice in images represented in (d). (n=3; 3 replicates) f, Appearance of GFP labeled E. coli (SWW33) exposed to buffer (control) or HMGB1. (n=3; 3 replicates) g, Flow cytometry of aggregates in samples of GFP labeled E. coli (SWW33) exposed to buffer (control) or HMGB1. (n=3; 3 replicates) h, Appearance of SYTO 9 labeled microbiota from C57BL/6 mice exposed to buffer (control) or HMGB1 labeled with AF647. Scale bars, 20 µm (n=3; 3 replicates) Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one individual mouse. Scale bars, 100 µm and original magnification 400x, unless otherwise noted.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Fluorescence, In Situ Hybridization, Real-time Polymerase Chain Reaction, Labeling, Isolation, Control, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Quantitative reverse transcriptase (qRT) PCR for expression of Mucin1 (Muc1) , Mucin2 (Muc2) , and Mucin3 (Muc3) in colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=9) b, Immunostaining for Mucin 2 (Muc2) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. (n=4) c, Lectin staining with fluorescently labeled Ulex europaeus agglutinin-1 (UEA-1) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Arrows indicate the epithelial surface. (n=4) Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one individual mouse. Scale bars, 100 µm. Original magnification 400x.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Immunostaining, Staining, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunostaining for HMGB1 in Carnoy’s fixed proximal colon sections from HMGB1 WT mice. Focal plane optimized to capture gut microbes. Scale bars, 100 µm. Original magnification 400x. Arrows indicate the leading edge of gut microbes. (n=20) b, Shannon alpha-diversity index of ASV abundances using DNA isolated from mucosal scrapings of colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) c, Canonical correspondence analysis (CCA) on ASV abundances using DNA isolated from mucosal scrapings of colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) d, Dimensional reduction plots used to characterize microbiome differences between the indicated sites (stool and mucosal) in samples from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) R 2 derived from permutational multivariate analysis of variance with site as the main variable. R 2 indicates the difference between the composition of the microbiota at the two sites in mice of each genotype (HMGB1 WT and HMGB1 ΔIEC ) and p-value indicates the significance of the difference in composition between the two sites. (n=12 WT/18 ΔIEC) e, Mean proportion of statistically different bacterial strains using DNA isolated from mucosal scrapings of colons from HMGB1 WT and HMGB1 ΔIEC mice. (n=12 WT/18 ΔIEC) Each datapoint represents one individual mouse except in (d) where each mouse has one datapoint for stool and one for mucosal sample.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Amino acid sequence similarities among the known human HMGB1 target proteins Beclin-1 and Atg5 and bacterial FimH. The putative HMGB1 interaction motif was derived from the amino acid sequence similarities between Beclin-1 and Atg5 and common amino acid replacements. b,c, Flow cytometry of rHMGB1 binding to E. coli (BW25113) knocked out for FimH (ΔFimH) or ΔFimH E. coli complemented with plasmids encoding wild type FimH (ΔFimH WT ; TSETPRV) or FimH mutated in the conserved residues of the putative motif (ΔFimH MUT ; ASATARA). Both the percent E. coli positive for HMGB1 (b) and the amount of HMGB1 protein (mean fluorescence intensity (MFI)) bound to each bacterium (c) were assessed. (n=3; 3 replicates) d, Label transfer of Sulfo-SBED from HMGB1 to recombinant FimH lectin domain (FimH LD ). Recipient proteins were wild type FimH LD (WT; TSETPRV) or FimH LD mutated in the conserved amino acid residues (mutant; ASATARA) of the putative interaction motif. Transfer was assessed in the absence or presence of mannose. (3 replicates) e, E. coli colony forming units (CFU) adherent to Caco2 IEC as a percent of input. E. coli were treated with buffer, rHMGB1, or mannose prior to addition to the IEC. (n=6; 3 replicates) f, Percentage of FimH LD protein bound to a mannose coated plate in the presence of increasing amounts of rHMGB1. (n=3; 3 replicates) g, RBC agglutination by E. coli (SWW33) expressing wild type FimH (FimH:TSETPRV), knocked out for FimH (FimH: KO), or expressing FimH mutated in the ToH1 sequence (FimH:ASATARA and FimH:AAAAAAA). Numbers of bacteria decrease from left to right. (3 replicates) h, Immunoblotting for FimH in mucus isolated from HMGB1 WT and HMGB1 ΔIEC mice. (n=4) i, Quantification of band densitometry of immunoblots represented in (h). (n=4) j, Immunostaining for FimH (red) and Hoescht (blue) in Carnoy’s fixed proximal colon sections from HMGB1 WT and HMGB1 ΔIEC mice. Scale bars, 100 µm. Original magnification 400x. (n=15) k, Quantification of FimH positive bacteria in images represented in (j). l, Immunoblotting for FimH in E. coli (SWW33) exposed to increasing amounts of HMGB1. (3 replicates) m, PCR determination of the orientation of the DNA switch region govering Fim gene expression in E. coli (ΔFimE) treated with media conditioned by IEC organoids derived from HMGB1 ΔIEC mice (ΔIEC CM), HMGB1 WT mice (WT CM), or ΔIEC CM supplemented with rHMGB1. Phase-on denotes switch oriented toward production of Fim genes. ftsZ is used for normalization. n, Relative band density of phase-on in (l). Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests for pairwise comparisons or one-way ANOVA with a Tukey post hoc test. Each datapoint represents one individual mouse.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Sequencing, Derivative Assay, Flow Cytometry, Binding Assay, Fluorescence, Recombinant, Mutagenesis, Agglutination, Expressing, Bacteria, Western Blot, Isolation, Immunostaining, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunofluorescence staining of HMGB1 (red) bound to E. coli (BW25113) (green). Wild type E. coli (WT), E. coli knocked out for FimH (ΔFimH), or ΔFimH E. coli complemented with plasmids carrying either WT FimH (ΔFimH WT ) or FimH mutated in ToH1 (ΔFimH Mut ) exposed to rHMGB1 and SYTO 9 to label bacterial DNA. b, Flow cytometry for FimH expression on the surface of ΔFimH E. coli complemented with plasmids carrying either WT FimH (ΔFimH WT ) or FimH mutated in ToH1 (ΔFimH Mut ). c, Flow cytometry gating strategy for HMGB1 biding reported in d, Flow cytometry of rHMGB1 binding to E. coli of the indicated strains. e, Flow cytometry gating strategy for HMGB1 binding reported in (d). Data are mean ± s.d. Significance determined by Student’s two-tailed T-tests. Each datapoint represents one biological replicate.

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Expressing, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: HMGB1 acts as an agent of host defense at the gut mucosal barrier

doi: 10.1101/2023.05.30.542477

Figure Lengend Snippet: a, Immunostaining for HMGB1 (yellow) and Hoescht (blue) in Carnoy’s fixed sections of resected colon from Non-IBD or UC patients. (n=16) b, Quantification of surface associated HMGB1 using images represented in (a). Staining intensity reported as relative fluorescent units (RFU) per μm 2 . (n=16) c, Surface associated HMGB1 reported in (b) graphed by inflammation severity. d, Immunostaining for FimH (red) and Hoescht (blue) in Carnoy’s fixed sections of resected colon from Non-IBD or UC patients. Serial tissue sections from the same patients represented in (a) (n=16) e, Quantification of FimH positive bacteria using images represented in (d). Reported as number of objects per high powered field. (n=16) f, Quantification of FimH positive bacteria reported in (e) graphed by inflammation severity. g, Surface HMGB1 and FimH positive bacteria plotted for each patient. The size of the closed circles corresponds to inflammation severity. Open ovals denote the population characteristics by group (non-IBD and UC). h, Two-way scatter plot of surface HMGB1 and FimH positive bacteria in each patient with a fitted curve. The relationship between HMGB1 and FimH was captured by non-linear regression. Data are mean ± s.d. Scale bars, 100 µm. Original magnification 400x. Each datapoint represents one individual person. Mann-Whitney U tests were used to compare HMGB1 and FimH in non-IBD vs. UC groups (two-group comparison), and Kruskal Wallis tests were used to assess the difference in HMGB1 and FimH among inflammation groups (three-group comparison).

Article Snippet: Samples were analyzed using an HMGB1 detection kit per the manufacturer’s instructions (Chondrex).

Techniques: Immunostaining, Staining, Bacteria, MANN-WHITNEY, Comparison

Journal: Neuron

Article Title: TREM2 is a receptor for β-amyloid which mediates microglial function

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For Aβ detection, brain sections were immunostained with MOAB-2 (M-1586-100, Biosensis) followed by an anti-mouse HRP secondary antibody, and developed using a DAB substrate (Vector Laboratories).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Mutagenesis, Plasmid Preparation, Software