Journal: Frontiers in Pharmacology

Article Title: Mycobacterium tuberculosis infection drives osteoclast overactivation via α2,3-Sialylation to promote pathological bone destruction

doi: 10.3389/fphar.2026.1738896

Figure Lengend Snippet: α2,3-sialylation is required for BCG-induced osteoclast differentiation and activity (A) Comparison of differentially expressed genes (DEGs) between BCG-infected osteoclasts (RB) and uninfected controls (R). (n = 3 biological replicates per group, differential expression was defined as |FoldChange| > 2 and padj <0.05). (B) Volcano plot of DEGs highlighting genes related to sialic acid biosynthesis. (C) KEGG pathway enrichment analysis of upregulated DEGs in RB cells. (D,E) Immunofluorescence staining of α2,3-SA in mouse calvarial sections using MAL II lectin, with quantification of α2,3-SA fluorescence intensity (n = 5). Intensity density was normalized to the PBS group mean. (F) In vitro osteoclasts subjected to MAL II lectin staining and TRAP staining, with or without sialidase treatment to enzymatically remove α2,3-SA. (G) Quantification of α2,3-SA fluorescence intensity in cultured osteoclasts (n = 5). Intensity density was measured in cellular ROIs after background subtraction and normalized to the RANKL group mean. (H) Quantification of TRAP + multinucleated cells (≥3 nuclei) per field (randomly selected fields, fixed magnification) in vitro (n = 5). (I) mRNA expression levels of osteoclast differentiation markers ( Fos, Mmp9, Nfatc1, and Ocstamp ) in osteoclasts (n = 3). Data are presented as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test for two-group comparisons (E) and one-way ANOVA followed by Tukey’s post hoc test for three-group comparisons (G–I) .

Article Snippet: Biotin MAL-II , Vector laboratories , Cat# B-1265-1.

Techniques: Activity Assay, Comparison, Infection, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence, In Vitro, Cell Culture, Expressing, Two Tailed Test

Journal: mSystems

Article Title: Adaptation of Enterococcus faecalis to intestinal mucus revealed by a human colonic organoid model

doi: 10.1128/msystems.01304-25

Figure Lengend Snippet: Colon epithelial monolayers with histological signatures for studying Enterococcus faecalis mucosal colonization. ( A ) Schematic depicting the experimental setup. Human colon organoids grown in Matrigel were split into single cells and seeded on top of collagen-coated Transwell membranes. Once the epithelial monolayer formed, the cells were differentiated at the air-liquid interface. Transwells were used on day 7 after seeding. ( B ) Immunostaining images of differentiated organoid monolayers with MUC2 protein (yellow), nuclei stained with DAPI (blue), and mucus labeled using Jacalin-biotin and streptavidin-Cy5 (magenta). ( C ) Colony-forming units (CFU/mL) analysis of Ef growth in mucus 6 h after inoculation. Both time points represent the colony-forming units of bacteria present in the Transwell in four independent biological replicates. The horizontal black lines mark the mean. ( D ) Maximum intensity projection of Jacalin-labeled mucus (magenta) and Ef WT expressing pDasherGFP (green). ( E ) Ef WT expressing pDasherGFP (green) in the same z-plane as the Jacalin-labeled mucus (magenta) and does not colocalize with the colonoid epithelium labeled with CellMask-DeepRed (red). ( F ) Ef colony volume calculated from confocal images represented in panel C . A minimum of 148 clusters was quantified from each image of four independent biological replicates.

Article Snippet: Jacalin-biotin , Vector Laboratories , VC-B-1155-M005.

Techniques: Immunostaining, Staining, Labeling, Bacteria, Expressing

Journal: mSystems

Article Title: Adaptation of Enterococcus faecalis to intestinal mucus revealed by a human colonic organoid model

doi: 10.1128/msystems.01304-25

Figure Lengend Snippet: Glycosyltransferase BgsB in E. faecalis is essential for growth in colonic mucus. ( A ) Table of log2FC values calculated from the Tn-seq experiment for the glycosyltransferase bgsB and a biofilm-forming factor bph . ( B ) Representative images of Ef colonies in colonic mucus at 9 h of growth from three biological replicates. Mucus was labeled using Jacalin-biotin and streptavidin-Cy5. Wild-type Ef fluorescently expressed pDasher-GFP, and bgsB and bph mutants expressed tdTomato. ( C ) Colony-forming unit (CFU/mL) quantification of the deletion mutants grown in mucus compared with the wild-type Ef after 6 h of growth. Each dot is a biological replicate (at least three independent biological replicates for each mutant), and the horizontal black lines mark the mean. ( D ) Competition between WT (green) and mutant samples (orange) with over three biological replicates, where the WT vs bgsB::Tn is marked with a filled circle and WT vs Δ bph with an unfilled triangle. Volume ratio is calculated as a fraction of the mutant signal occupying the total (mutant + WT) volume in the image. Volume expansion is calculated as a ratio to the initial volume in the respective channel. Mean and standard deviation are shown. ( E ) The same mixed samples were grown in mucus in a flow chip shown in , under the flow of 5 µL/min of minimal medium ( n = 3). Arrows indicate the same place at the start and the end of the time-lapse. Statistics in panel C were calculated using one-way ANOVA with post hoc Dunnett’s multiple comparison test (*** P < 0.001).

Article Snippet: Jacalin-biotin , Vector Laboratories , VC-B-1155-M005.

Techniques: Labeling, Mutagenesis, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) SLC35A2 encodes a transporter for UDP-galactose, which is required for the synthesis of O-GalNAc glycans. B) The lectin VVA binds terminal alpha-GalNAc (Tn-antigen), while PNA binds terminal Gal of Core 1 O-GalNAc glycans (T-antigen), which can be removed by O-glycosidase (OGA). C) Breeding strategy of Emx1-Cre mice with floxed- Slc35a2 mice, with blue regions highlighting the forebrain expression Emx1 . D) VVA binding is minimal in protein blots of wild-type mouse cortex; female (+/+) and male (+/y) with floxed- Slc35a2 . In contrast, mice lacking Slc35a2 in cells expressing the forebrain-specific promoter Emx1 ( Emx1-Cre ) display robust VVA binding in both females (+/-) and males (-/y), consistent with inhibition of O-GalNAc extension. Binding of PNA after Neuraminidase A (NeuA) treatment was significantly reduced in both +/- and -/y lines, and specificity of PNA binding was confirmed via OGA treatment of a +/+ sample. ConA, which binds all N-glycans, was unaffected in all mouse lines, and specificity was confirmed using peptide N-glycosidase F (PNGase F) treatment of a +/+ sample. Each lane contains 15 µg cortical protein lysate from an individual mouse of the corresponding genotype. Experiment was replicated three times with similar results. E) Quantification of lectin binding from D normalized to total protein (TP) staining within the same lane of each blot. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse included, N = 3/genotype. One-way ANOVA confirmed significant group differences ( p < 0.05) for VVA and PNA but not ConA, followed by post hoc Student’s t-tests to confirm genotype effects in each sex: ** p -value = 0.01, **** p -value = 0.0001. F) Schematic of coronal mouse brain section highlighting the cortex (CTX), corpus callosum (CC), and basal ganglia (BG). G) VVA binding is minimal across all regions in wildtype mouse cortex (+/y), while PNA binding localizes to neuronal tracts of the CC and axon bundles in the BG. In contrast, mice lacking forebrain expression of Slc35a2 (-/y) display a build-up of truncated O-GalNAc glycans in cortex that fail to properly localize. Mature O-GalNAc glycans are entirely absent from the CTX and CC, but preserved in the BG, which does not express Emx1 -Cre. H) Relative quantification of lectin binding across brain regions (CTX, CC, BG) from G. Data presented as the mean binding intensity +/- SEM, with individual points representing the average of 6 measures across independent mice. Colored bars correspond with the specific brain region indicated in (F): CTX, purple; CC, orange; BG, blue. N = 2 for +/y VVA; N = 4 for +/- PNA, -/y VVA, and -/y PNA. Fluorescence was quantified as arbitrary intensity (a.i.) after subtracting background signal from each sample.

Article Snippet: Biotinylated lectins ConA (VectorLabs,#B-1005-5, 25 μg/mL) and VVA (VectorLabs, #B-1235-2, 25 μg/mL) were diluted in TBS-T and incubated for 1 hour at room temperature.

Techniques: Expressing, Binding Assay, Inhibition, Staining, Quantitative Proteomics, Fluorescence

Journal: European journal of immunology

Article Title: Endoglycosidase treatment abrogates IgG arthritogenicity: importance of IgG glycosylation in arthritis.

doi: 10.1002/eji.200737581

Figure Lengend Snippet: Figure 1. Analysis of EndoS-treated CII-specific mAb. SDS-PAGE and lectin blot analysis of IgG mAb (M2139 – IgG2b and CIIC1 – IgG2a) incubated with and without EndoS and separated by 10% SDS-PAGE. The proteins were detected by Coomassie Brilliant Blue staining (Stain) or by blotting onto a membrane that was probed with LCA lectin (Blot).

Article Snippet: The membranes were blocked with 10 mM HEPES (pH 7.5) with 0.15 M NaCl, 0.01 mM MnCl2, 0.1 mM CaCl2, and 0.1% Tween-20 (HBST) and incubated with 1 lg/mL biotinylated LCA lectin (Vector Laboratories, Burlingame, CA).

Techniques: SDS Page, Incubation, Staining, Membrane

Journal: European journal of immunology

Article Title: Endoglycosidase treatment abrogates IgG arthritogenicity: importance of IgG glycosylation in arthritis.

doi: 10.1002/eji.200737581

Figure Lengend Snippet: Figure 2. EndoS-treated Ab bind to cartilage in vivo. Neonatal rats 1–2 days old were injected with 1 mg of CII-binding Ab [both normal and EndoS-treated (D)]) i.p. At 24 h after the Ab transfer, paws were dissected and snap-frozen in OCT compound using isopentane and dry ice. Immunohistochem- ical analysis was performed using biotinylated anti-mouse kappa (187.1) Ab and streptavidin peroxidase as detecting system using standard protocols. Stained joint sections (10 lm) from neonatal rats injected with M2139 (A), M2139D (B), CIIC1 (C), CIIC1D (D) and control, untreated rats (E) are shown. Magnification 10.

Article Snippet: The membranes were blocked with 10 mM HEPES (pH 7.5) with 0.15 M NaCl, 0.01 mM MnCl2, 0.1 mM CaCl2, and 0.1% Tween-20 (HBST) and incubated with 1 lg/mL biotinylated LCA lectin (Vector Laboratories, Burlingame, CA).

Techniques: In Vivo, Injection, Binding Assay, Staining, Control