Journal: bioRxiv

Article Title: Sialidases derived from Gardnerella vaginalis remodel the sperm glycocalyx and impair sperm function

doi: 10.1101/2025.02.01.636076

Figure Lengend Snippet: A) Schematic describing lectin flow cytometry and representative glycan epitopes. B, C, D) Sperm were exposed to sialidases (AUS 20 units or GvNanH2 160 units) for 1 hour, fixed in formalin, and stained with MAL-II-biotin/Neutravidin-Alexa Fluor 488, SNA-Cy5, or PSA-FITC respectively. Each point represents the median fluorescence intensity from one donor. Statistics represent a mixed model comparison with post-hoc Holm-Šidák tests. E) Schematic representing change in zeta potential after removal of negatively charged sialic acid on sperm surface. F) Sperm were treated with sialidase at various doses for 1 hour, and zeta potential was measured via dynamic light scattering. Each measurement represents the average of seven technical replicates, and statistics represent a mixed model comparison with post hoc Holm-Šidák tests.

Article Snippet: Biotinylated Maackia Amurensis Lectin II (MAL II, Cat #:B-1265-1) and Cy5-conjugated Sambucus Nigra Lectin (SNA, Cat #: CL-1305-1) were purchased from Vector Labs. Human Contraception Antibody (HCA), was a gift from ZabBio.

Techniques: Flow Cytometry, Staining, Fluorescence, Comparison, Zeta Potential Analyzer

Journal: bioRxiv

Article Title: The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

doi: 10.1101/646794

Figure Lengend Snippet: (A) Human GALE epimerizes two pairs of NSs, UDP-Gal/UDP-Glc and UDP-GalNAc/UDP-GlcNAc. (B) GALE was deleted using CRISPR/Cas9 methods and one of three GALE-targeting sgRNAs (1-3) in 293T (left) and HeLa (right) cells. Single-cell derived clones (denoted A or B) were lysed and analyzed by Western blot. (C) Control and GALE −/− HeLa cells were treated with 250 μM Gal or mannitol (osmolarity control) for 72 hours and UDP-Gal and UDP-GalNAc were quantified by high performance anion exchange chromatography (HPAEC). N=3 biological replicates. Error bars represent standard error of the mean (SEM). See also . (D) Control and GALE −/− 293T (left) and HeLa (right) cells were stained with fluorescently (FITC) tagged jacalin, SNA or WGA lectins and 10,000 cells from each sample were analyzed by flow cytometry. The major glycan ligand of each lectin is indicated between the corresponding panels.

Article Snippet: The lectins used were fluorescein-SNA (vector labs FL-1301-2), fluorescein-WGA (vector labs FL-1201) and fluorescein-jacalin (vector labs FL-1121).

Techniques: CRISPR, Derivative Assay, Clone Assay, Western Blot, Chromatography, Staining, Flow Cytometry

Journal: Cancers

Article Title: Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity

doi: 10.3390/cancers14030603

Figure Lengend Snippet: Differential binding of lectins to breast cancer cell lines. ( A , B ) The parental cells MB-231 and its bone-seeking derivatives, MET and BONE, were cell-surface labeled with one of a panel of distinct FITC-conjugated lectins prior to cytochemistry (( A ), left panels) and flow cytometry (( A ), right panels, ( B )) analyses. See also . Percentages of positive cells are indicated in the histograms ( A ) and the median fluorescence intensity is presented for each lectin ( B ). Data from representative experiments acquired under uniform instruments setting for all cell lines are displayed. The lectins used were Concanavalin A (ConA), Datura Stramonium Lectin (DSL), Dolichos Biflorus Agglutinin (DBA), Erythrina Cristagalli Lectin (ECL), Griffonia Simplicifolia Lectin I (GSL-I), Griffonia Simplicifolia Lectin II (GSL-II), Lens Culinaris Agglutinin (LCA), Lycopersicon Esculentum Lectin (LEL), Peanut Agglutinin (PNA), Phaseolus Vulgaris Lectin E (PHA-E), Phaseolus Vulgaris Lectin L (PHA-L), Pisum Sativum Agglutinin (PSA), Ricinus Communis Agglutinin I (RCA), Sambucus Nigra Lectin (SNA), Solanum Tuberosum Lectin (STL), Soybean Agglutinin (SBA), Ulex Europaeus Agglutinin I (UEA-I), Vicia Villosa Lectin (VVL) and Wheat Germ Agglutinin (WGA, succinylated (succ)-WGA). Scale bar, 25 μm.

Article Snippet: After inactivation of trypsin, 2 washing steps with PBS and centrifugation (5 min at 300× g ), cells were resuspended in PBS or Ca/Mg-PBS (for lectin labeling), containing 1% BSA and 100 μL-cell suspension aliquots were incubated with unconjugated or fluorochrome-conjugated primary antibodies ( ) or FITC- or biotin-conjugated lectins (see above, Lectin kit I, Biotinylated (BK-1000) and SNA, Biotinylated (B-1305-2), both from Vector Laboratories) for 30 min at 4 °C.

Techniques: Binding Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and sialic acid linkage in S. suis serotype 2 and 14 mutants carrying exogenous α-2,3-sialyltransferase. (A) Hydrophobicity (%) of the wild-type S. suis serotype 2 (SS2) and 14 (SS14) strains, the SS2sia2,3 (Δ cps2N / cpsK ) and SS14sia2,3 (Δ cps14N / cpsK ) mutants carrying the GBS α-2,3-sialyltransferase ( cpsK ). The non-encapsulated mutants SS2Δ cps and SS14Δ cps were used as control strains. (B,C) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in SS2sia2,3 and SS14sia2,3 mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutants SS2Δ cps and SS14Δ cps were used as negative controls. SS2 was used as positive control for SNA-I and wild-type GBS type III as positive control for MAL-I. Data in (A–C) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation, Positive Control

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and recognition of specific CPS sialic acid linkage in GBS type V isogenic mutants. (A) Hydrophobicity (%) of the wild-type GBS serotype V strain (GBS V), and the sialic acid synthesis GBSVΔsynth (Δ neu5B ) and sialyltransferase GBSVΔsiaT (Δ cps5K ) deficient mutants. The non-encapsulated strain (GBSVΔ cps ) was used as control. (B) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in these mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutant GBSVΔ cps was used as negative control. S. suis serotype 2 (SS2) was used as positive control for SNA-I and wild-type GBS type V as positive control for MAL-I. Data in (A,B) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation, Negative Control, Positive Control

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and sialic acid linkage in GBS type III and V mutants carrying exogenous α-2,6-sialyltransferase. (A) Hydrophobicity (%) of GBS type III and V wild-type strains and the mutants GBSIIIsia2,6 (Δ cps3K / cps2N ) and GBSVsia2,6 (Δ cps5K / cps2N ) carrying the S. suis α-2,6-sialyltransferase. The non-encapsulated mutant strains (GBSIIIΔ cps and GBSVΔ cps ) were used as controls. (B,C) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in these mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutants were used as negative controls. Data in (A–C) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” , between “ a” and “ c” , and between “ b” and “ c” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation