anti mouse cf 488a  (Biotium)

Journal: Pharmaceutics

Article Title: Functional and Safety Profile of Limosilactobacillus vaginalis and Development of Oral Fast-Disintegrating Tablets for Gut Microbiota Modulation

doi: 10.3390/pharmaceutics17081011

Figure Lengend Snippet: Effect of L. vaginalis BC17 fractions (viable BC17, CFS-BC17, HK-BC17, viable+CFS-BC17 and HK+CFS-BC17) on Bifidobacterium spp. adhesion to Caco-2 cells. ( A ) Adherent Bifidobacterium per Caco-2 cell after 3 h of co-incubation (mean ± SD, n = 3), * p < 0.05 (vs. control). ( B ) Boxplot of Bifidobacterium adhesion percentage compared to the control (100%). Each box represents the interquartile range (25–75th percentile). Lines within the boxes indicate the median values of the samples. The extremes of the bars indicate the minimum and maximum values, respectively. Statistically significant differences between the samples are indicated by different letters ( p < 0.05).

Article Snippet: Bifidobacterium strains ( B. breve DSM 20091, B. breve DSM 20456, B. bifidum DSM 20082, B. bifidum DSM 20213, B. bifidum DSM 20215, B. longum subsp. longum DSM 20219, B. longum subsp. infantis DSM 20088, B. longum subsp. infantis DSM 20090, B. adolescentis DSM 20083, B. adolescentis DSM 20086, B. angulatum DSM 20098) were purchased from DSMZ [ ].

Techniques: Incubation, Control

Journal: Pharmaceutics

Article Title: Functional and Safety Profile of Limosilactobacillus vaginalis and Development of Oral Fast-Disintegrating Tablets for Gut Microbiota Modulation

doi: 10.3390/pharmaceutics17081011

Figure Lengend Snippet: Functional characterization of tablets (Tab HK+CFS-BC17 and Tab viable+CFS-BC17). Tablets were tested immediately after the preparation (T0) and after 3 months of storage at room temperature (T3). ( A ) Effects on viability of Caco-2 and HT-29 cells after 24 h and 48 h of incubation, evaluated by MTT assay and dye exclusion test. Overall results are presented as boxplot reporting the increase of cell viability (%) compared to the control (dotted line). ( B ) Effects on Bifidobacterium spp. adhesion to Caco-2 cells. Boxplot reported the percentages compared to the control (100%). ( C ) Protective effects on Caco-2 and HT-29 cells exposed to SDS 0.05% for 24 h and 48 h. Overall results are presented as boxplot reporting the increase of cell viability (%) compared to cells exposed to SDS (dotted line). Each box represents the interquartile range (25–75th percentile). Lines within the boxes indicate the median values of the samples. The extremes of the bars indicate the minimum and maximum values, respectively. ( D ) Inhibitory effect on NO production in LPS-induced macrophages. Results are in percentages with respect to control (means ± SD). All tests were performed in triplicate; statistically significant differences between the samples are indicated by different letters ( p < 0.05).

Article Snippet: Bifidobacterium strains ( B. breve DSM 20091, B. breve DSM 20456, B. bifidum DSM 20082, B. bifidum DSM 20213, B. bifidum DSM 20215, B. longum subsp. longum DSM 20219, B. longum subsp. infantis DSM 20088, B. longum subsp. infantis DSM 20090, B. adolescentis DSM 20083, B. adolescentis DSM 20086, B. angulatum DSM 20098) were purchased from DSMZ [ ].

Techniques: Functional Assay, Incubation, MTT Assay, Control

Journal: Pharmaceutics

Article Title: Functional and Safety Profile of Limosilactobacillus vaginalis and Development of Oral Fast-Disintegrating Tablets for Gut Microbiota Modulation

doi: 10.3390/pharmaceutics17081011

Figure Lengend Snippet: L. vaginalis BC17 adhesion to Caco-2 cells co-incubated with Bifidobacterium spp. and enteropathogens. The adhesion of L. vaginalis BC17 without test microorganisms (BC17 alone) was evaluated for comparison as follows: viable BC17 at ratios of 100:1 and 33:1 with Caco-2 cells, and viable BC17 in the presence of CFS (viable+CFS-BC17). ( A ) Results are reported as adherent BC17 per Caco-2 cell for L. vaginalis in the presence of bifidobacteria, enterotoxigenic E. coli (ETEC), S. enterica or Y. enterocolitica (mean ± SD, n = 3), * p < 0.05 (viable BC17+ test microorganisms vs. BC17 alone 100:1), # p < 0.05 (viable+CFS-BC17 + test microorganisms vs. viable+CFS-BC17 alone). ( B ) Boxplot of adherent BC17 per Caco-2 cell. Lines within the boxes indicate the median values of the samples. The extremes of the bars indicate the minimum and maximum values, respectively. Statistically significant differences between the samples are indicated by different letters ( p < 0.05).

Article Snippet: Bifidobacterium strains ( B. breve DSM 20091, B. breve DSM 20456, B. bifidum DSM 20082, B. bifidum DSM 20213, B. bifidum DSM 20215, B. longum subsp. longum DSM 20219, B. longum subsp. infantis DSM 20088, B. longum subsp. infantis DSM 20090, B. adolescentis DSM 20083, B. adolescentis DSM 20086, B. angulatum DSM 20098) were purchased from DSMZ [ ].

Techniques: Incubation, Comparison

Journal: Experimental and molecular pathology

Article Title: Cross-feeding between beneficial and pathogenic bacteria to utilize eukaryotic host cell-derived sialic acids and bacteriophages shape the pathogen-host interface milieu.

doi: 10.1016/j.yexmp.2025.104967

Figure Lengend Snippet: Fig. 1. Cell viability assay. The impact of P.a PAO1, KPP22 phage, and B. bifidum on the cell viability of T84 cells (panel A), THP-1 cells (panel B), and Huh7 cells (panel C) is shown. The seeded triple co-cultured cells were incubated in DMEM culture medium (white bars) or DMEM culture medium with 100 μM of DANA (black bars). The cells were then exposed for 6 h to the following treatments: 1) P.a PAO1 (1 × 108CFU/ml) alone; 2) P.a PAO1 plus its KPP22 phage; and 3) P.a PAO1 plus B. bifidum (1 × 108CFU/ml). In the control group, cells were incubated with only medium. The KPP22 phage was used as a specific natural phage lysing P.a PAO1. DANA was used as a specific endogenous sialidase inhibitor. The mean values of the control wells containing only cells without any bacteria were taken as 100 %. The percentage of viable active cells treated with bacteria, phage, or DANA was then calculated. Each bar represents the mean of three independent replicates (n = 3) with its corresponding standard error of the mean (SEM). Statistical comparisons were conducted using a one-way ANOVA with Tukey’s post-test (A, B, and C). Data are expressed as percentage (%) cell survival compared to the control. The bars with different lowercase letters (a-g) indicate statistically significant differences between any two groups (p < 0.05), indicating that they are significantly different from each other. Bars with the same lowercase letters represent no significant differences between any two groups (p < 0.05). P.a PAO1, Pseudomonas aeruginosa PAO1; B.bifidum, Bifidobacterium bifidum; KPP22, KPP22 phage.

Article Snippet: Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bifidobacterium bifidum DSM 20456 were purchased from DSMZ (Braunschweig, Germany).

Techniques: Viability Assay, Cell Culture, Incubation, Control, Bacteria

Journal: Experimental and molecular pathology

Article Title: Cross-feeding between beneficial and pathogenic bacteria to utilize eukaryotic host cell-derived sialic acids and bacteriophages shape the pathogen-host interface milieu.

doi: 10.1016/j.yexmp.2025.104967

Figure Lengend Snippet: Fig. 2. Transepithelial electrical resistance (TEER) of T84 cell monolayers. The monolayers of T84 cells grown on transwell inserts were exposed to P.a PAO1 (1 × 108 CFU/ml) and B. bifidum (1 × 108 CFU/ml) for 6 h. The error bars indicate the standard deviation of at least three independent experiments. Data are expressed as means ±SEM. Bars with different lowercase letters represent statistically significant differences between any two groups (p < 0.05), indi cating that they are significantly different from each other. The bars with common (same) lowercase letters represent no significant differences between any two groups (p < 0.05).

Article Snippet: Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bifidobacterium bifidum DSM 20456 were purchased from DSMZ (Braunschweig, Germany).

Techniques: Standard Deviation

Journal: Experimental and molecular pathology

Article Title: Cross-feeding between beneficial and pathogenic bacteria to utilize eukaryotic host cell-derived sialic acids and bacteriophages shape the pathogen-host interface milieu.

doi: 10.1016/j.yexmp.2025.104967

Figure Lengend Snippet: Fig. 3. Release of free sialic acid from the surface of co-cultured cells and sialidase activity in the cell culture supernatant. T84, THP-1, and Huh7 cells were co-cultured in DMEM medium or DMEM + DANA and incubated with P.a PAO1, P.a PAO1+ KPP22 phage or P.a PAO1+ B. bifidum for 6 h. The control group was incubated with only medium for comparison. (A) The amount of free sialic acid remaining in the cell culture supernatants 6 h after inoculation of P.a PAO1 and B. bifidum or uninoculated media control. (B) Sialidase activity was measured in 6-h co-cultures using supernatants from panel A. After 6 h of in cubation, the activity of sialidase in the supernatants was analyzed by spec trophotometry and expressed as absorbance (Ab) at 595 nm. Both panels show the mean values of three independent experiments, with standard errors of the mean (± SEM). The bars with different lowercase letters (a-g) represent sta tistically significant differences between any two groups (p < 0.05), indicating that they differ significantly from each other. The bars with common (same) lowercase letters represent no significant differences between any two groups (p < 0.05). The bars represent the mean values of three independent experi ments carried out in duplicate.

Article Snippet: Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bifidobacterium bifidum DSM 20456 were purchased from DSMZ (Braunschweig, Germany).

Techniques: Cell Culture, Activity Assay, Incubation, Control, Comparison

Journal: Experimental and molecular pathology

Article Title: Cross-feeding between beneficial and pathogenic bacteria to utilize eukaryotic host cell-derived sialic acids and bacteriophages shape the pathogen-host interface milieu.

doi: 10.1016/j.yexmp.2025.104967

Figure Lengend Snippet: Fig. 4. Influence of DANA, the KPP22 phage, and B. bifidum on the host- pathogen interaction of P.a PAO1 with T84 monolayer cells. The effects of KPP22 phage (1 × 108 PFU/ml), B. bifidum (1 × 10 8 PFU/ml), and the sialidase inhibitor DANA on P.a PAO1 (1 × 108CFU/ml) adhesion to (A) and invasion into (B) human T84 intestinal epithelial cells after 6h of infection. The error bars indicate the standard deviation of at least three independent experiments. Data are expressed as means ±SEM. The bars with different lowercase letters represent statistically significant differences between any two groups (p < 0.05), indicating that they differ significantly from each other. The bars with common (same) lowercase letters represent no significant differences between any two groups (p < 0.05).

Article Snippet: Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bifidobacterium bifidum DSM 20456 were purchased from DSMZ (Braunschweig, Germany).

Techniques: Infection, Standard Deviation

Journal: Experimental and molecular pathology

Article Title: Cross-feeding between beneficial and pathogenic bacteria to utilize eukaryotic host cell-derived sialic acids and bacteriophages shape the pathogen-host interface milieu.

doi: 10.1016/j.yexmp.2025.104967

Figure Lengend Snippet: Fig. 5. Effects of DANA, KPP22 phage, and B. bifidum on PAO1-induced secretion of pro-inflammatory cytokines from triple co-cultured cells. The seeded triple co-cultured cells were incubated in DMEM culture medium (white bars) or DMEM culture medium with 100 μM of DANA (black bars). The upper medium of the co-culture system was treated with indicated concentrations of PAO1 (1 × 108 CFU/ml), KPP22 phage (1 × 108 PFU/ml), and B. bifidum (1 × 108 CFU/ml) for 6 h. In the control group, cells were incubated with only medium. The secretion of IL-6 (A) and IL-8 (B) from the co-cultured cells was determined by an ELISA assay. Bars with different lowercase letters represent statistically significant differences between any two groups (p < 0.05), indi cating that they are significantly different from each other. The bars with common (same) lowercase letters represent no significant differences between any two groups (p < 0.05). Each value is presented as the mean ± SEM (n = 3).

Article Snippet: Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bifidobacterium bifidum DSM 20456 were purchased from DSMZ (Braunschweig, Germany).

Techniques: Cell Culture, Incubation, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 1. Probiotic Bifidobacterium bifidum strains uniquely modify the MUC13 extracellular domain. (A) Immunoblot analysis of MUC13 modification/degradation and claudin-3 expression in HRT18 monolayers after incubation with different probiotic strains at MOI 10 for 20 h. The full-length 130 kDa MUC13- band that is used for quantification in (B) is marked by an arrow. (B,C) Quantification of protein expression of the 130 kDa MUC13 and 22 kDa claudin-3 bands (relative to uninfected control) in three biological replicates as depicted in A. All immunoblots used for quantification are available in Figure S1 and Figure S2. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (D) Fluorescence confocal microscopy images of HRT18 cells stained in green with MUC13-ED (above) and MUC13-CT (below) antibodies after 20 h incubation with B. bifidum strains at MOI 10 and StcE. The maximal intensity projection is depicted. White scale bars represent 20 µM. (E) Quantification of GFP signal in cells stained with MUC13-ED and MUC13-CT antibodies as in (C). Statistical analysis was performed on non-normalized data using a one-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. (F) Immunoblot analysis of degradation of high-MW MUC13 (130 kDa) after 2 and 20 h incubation with B. bifidum W23, W28, and 20456 strains at MOI 10 and 50 in HRT18 cells. The full-length 130 kDa MUC13- band that is used for quantification in (G) is marked by an arrow. (G) Quantification of protein expression of MUC13 (relative to uninfected control) in three biological replicates as depicted in F. All immunoblots used for analysis are available in Figure S3. Statistical analysis was performed on non-normalized data using a one-way ANOVA or Krustal-Wallis test (when the data was not normally distributed) with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. Bacteria are listed in Table 1. All graphs depict the SEM of three independent experiments.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Western Blot, Modification, Expressing, Incubation, Control, Fluorescence, Confocal Microscopy, Staining, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 2. Bifidobacterium bifidum strains W23 and W28 have high sialidase activity that results in desialylation of surface proteins including MUC13. (A) Fucosidase and (B) sialidase activities in bacterial pellets of the B. bifidum strains. Graphs depict experimental results of three independent biological replicates. The commercial α1,2,3,4,6-L-fucosidase (Megazyme, E-FUCHS) was used as a positive control for fucosidase activity. All graphs depict the SEM of three independent experiments. Statistical test: two-tailed independent t-test. **** p < 0.0001. (C) Number of identified O-glycan-targeting CAZymes in the genome of B. bifidum strains 20456. (D) Western blot analysis of sialic acid-containing proteins in the HRT18-∆MUC13 + pMUC13 cell line after 2 h incubation with UV-inactivated B. bifidum strains at MOI 50 probes with lectins SNA (α-2,6 sialic acids) and MAL-II (α-2,3 sialic acids) lectins. Red arrows mark evidently reduced bands after incubation with W23 and W28 compared to the other lanes. Similar results were obtained in two independent experiments. (E) Anti-GFP immunoprecipitation and immunoblot analysis of MUC13-GFP (~ 160 kDa) from HRT18- ∆MUC13 + pMUC13 cells line after incubation with UV-inactivated B. bifidum at MOI 50 for 2 h. (F) Anti- GFP immunoprecipitation and immunoblot analysis of MUC13-GFP with MAL-II lectin after incubation with UV-inactivated B. bifidum strains at MOI 200 for 5 h. All immunoblots generated for experiments depicted in (D–F) analysis are available in Figure S4. (G) Immunoblot analysis of MUC13-GFP and actin from HRT18-∆MUC13 + pMUC13 cells incubated with UV-inactivated W28 bacteria at MOI 50 for 4 h. Conditions also included addition of 1.5 mM sialidase inhibitor (DANA) or 1 × Halt protease and phosphatase inhibitor cocktail (p.i.). Cells were also treated with a combination of 200 U/mL of α2,3,6,8,9 neuraminidase A and 0.6 U of α1,2,3,4,6-L-fucosidase. (H) Immunoblot analysis of MUC13-GFP and actin from HRT18- ∆MUC13 + pMUC13 cells incubated with UV-inactivated W28 bacteria at MOI 120 for 5 h in the absence or presence of 5.5 mM DANA. All immunoblots generated for experiments depicted in (G,H) are available in Figure S5.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Activity Assay, Positive Control, Two Tailed Test, Glycoproteomics, Western Blot, Incubation, Immunoprecipitation, Generated, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 3. Adherence of Bifidobacterium bifidum strains to HRT18 monolayers and penetration of the secreted mucus layer in Caco-2 ALI/VIP cultures. (A) FISH staining in combination with confocal microscopy of UV-killed B. bifidum adhesion to HRT18 monolayers at MOI 10 and 50 for 20 h using a 16S PNA probe (green). White scale bars represent 20 µM. (B) Adhesion of B. bifidum W23, W28, and 20456 strains to HRT18 monolayers assessed by quantification of colony-forming units (CFUs). The graph represents the average and SEM of three independent experiments. Statistical test: one-way ANOVA with Tukey’s correction. (C) Schematic representation of Caco-2 cells cultured in Transwells under liquid–liquid interface (LLI) and air– liquid interface (ALI) with the basolateral addition of vasointestinal peptide (VIP). Caco-2 ALI/VIP cultures produce a secreted mucus layer on the apical surface41 (D) Fluorescence confocal microscopy images of Caco-2 cells grown under ALI/VIP conditions to induce mucus formation incubated with pre-stained W23, W28, 20456, and W1 bacteria (green) at MOI 50 for 4 h. The secreted mucus layer was stained with the lectin Jacalin (orange) and DAPI was used for the nuclei (blue). The middle of the image shows the maximal intensity projection of the mucus and bacterial stainings, while the orthogonal view depicts single planes for clarity. White scale bars represent 20 µM. (E) 3D rendering of images is shown in (B). (F) Immunoblot analysis of MUC13 modification in the Caco-2 ALI/VIP cultures after incubation with B. bifidum strains at MOI 50 for 4 h. All immunoblots used for quantification are available in Figure S6. (G) Quantification of protein abundance of the high and low MW MUC13 (relative to actin) as shown in (D). Graph depicts experimental results of three independent biological replicates. Statistical test: one-way ANOVA with Dunnett’s post hoc test. * p < 0.05.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Staining, Confocal Microscopy, Cell Culture, Fluorescence, Incubation, Bacteria, Western Blot, Modification, Quantitative Proteomics

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 4. Bifidobacterium bifidum strains W23 and W28 enhance intestinal barrier properties. (A) Immunofluorescence confocal images of HRT18 cells grown in glass slides until full confluency and infected with B. bifidum strains W23, W28, and 20456 at MOI 10 for 20 h. Cells were stained for occludin (green), ZO-1 (red), and DAPI (white). The maximum intensity projection is depicted. White scale bars represent 20 µM. (B) Immunofluorescence images of HRT18 cultures grown in Transwells incubated with W23, W28, 20456, W1, and W37 at MOI 100 for 28 h. Cultures were stained for occludin (green), ZO-1 (red), and DAPI (white). Z-stacks were captured from the apical surface until the appearance of nuclei and maximum intensity projections are depicted. White scale bars represent 20 µM. (C) Immunoblot analysis of MUC13 modification and expression of claudins-1, -3, and -4 in HRT18 Transwell cultures after incubation with bacterial strains W23, W28, 20456, W1, and W37 at MOI 100 for 28 h and during a challenge with 100 ng/mL IL-1β and 100 ng/mL TNF-α. The control sample only received the cytokine challenge without bacteria. All immunoblots used for quantification are available in Figure S7. (D) Quantification of protein expression of MUC13 and claudins-1, -3, and -4 (relative to actin) as shown in (C). All graphs depict the SEM of three independent experiments. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (E) TEER measurements of HRT18 Transwell cultures over time during incubation with W23, W28, 20456, W1, and W37 strains at MOI 100 and cytokine challenge at baseline as described for C. (F) TEER values at the 28 h timepoint depicted in E. Graphs depict SEM of three independent biological replicates. Statistical test: two-way ANOVA with Dunnett’s post hoc test. **** p < 0.0001.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Immunofluorescence, Infection, Staining, Incubation, Western Blot, Modification, Expressing, Control, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 1. Probiotic Bifidobacterium bifidum strains uniquely modify the MUC13 extracellular domain. (A) Immunoblot analysis of MUC13 modification/degradation and claudin-3 expression in HRT18 monolayers after incubation with different probiotic strains at MOI 10 for 20 h. The full-length 130 kDa MUC13- band that is used for quantification in (B) is marked by an arrow. (B,C) Quantification of protein expression of the 130 kDa MUC13 and 22 kDa claudin-3 bands (relative to uninfected control) in three biological replicates as depicted in A. All immunoblots used for quantification are available in Figure S1 and Figure S2. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (D) Fluorescence confocal microscopy images of HRT18 cells stained in green with MUC13-ED (above) and MUC13-CT (below) antibodies after 20 h incubation with B. bifidum strains at MOI 10 and StcE. The maximal intensity projection is depicted. White scale bars represent 20 µM. (E) Quantification of GFP signal in cells stained with MUC13-ED and MUC13-CT antibodies as in (C). Statistical analysis was performed on non-normalized data using a one-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. (F) Immunoblot analysis of degradation of high-MW MUC13 (130 kDa) after 2 and 20 h incubation with B. bifidum W23, W28, and 20456 strains at MOI 10 and 50 in HRT18 cells. The full-length 130 kDa MUC13- band that is used for quantification in (G) is marked by an arrow. (G) Quantification of protein expression of MUC13 (relative to uninfected control) in three biological replicates as depicted in F. All immunoblots used for analysis are available in Figure S3. Statistical analysis was performed on non-normalized data using a one-way ANOVA or Krustal-Wallis test (when the data was not normally distributed) with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. Bacteria are listed in Table 1. All graphs depict the SEM of three independent experiments.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Western Blot, Modification, Expressing, Incubation, Control, Fluorescence, Confocal Microscopy, Staining, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 2. Bifidobacterium bifidum strains W23 and W28 have high sialidase activity that results in desialylation of surface proteins including MUC13. (A) Fucosidase and (B) sialidase activities in bacterial pellets of the B. bifidum strains. Graphs depict experimental results of three independent biological replicates. The commercial α1,2,3,4,6-L-fucosidase (Megazyme, E-FUCHS) was used as a positive control for fucosidase activity. All graphs depict the SEM of three independent experiments. Statistical test: two-tailed independent t-test. **** p < 0.0001. (C) Number of identified O-glycan-targeting CAZymes in the genome of B. bifidum strains 20456. (D) Western blot analysis of sialic acid-containing proteins in the HRT18-∆MUC13 + pMUC13 cell line after 2 h incubation with UV-inactivated B. bifidum strains at MOI 50 probes with lectins SNA (α-2,6 sialic acids) and MAL-II (α-2,3 sialic acids) lectins. Red arrows mark evidently reduced bands after incubation with W23 and W28 compared to the other lanes. Similar results were obtained in two independent experiments. (E) Anti-GFP immunoprecipitation and immunoblot analysis of MUC13-GFP (~ 160 kDa) from HRT18- ∆MUC13 + pMUC13 cells line after incubation with UV-inactivated B. bifidum at MOI 50 for 2 h. (F) Anti- GFP immunoprecipitation and immunoblot analysis of MUC13-GFP with MAL-II lectin after incubation with UV-inactivated B. bifidum strains at MOI 200 for 5 h. All immunoblots generated for experiments depicted in (D–F) analysis are available in Figure S4. (G) Immunoblot analysis of MUC13-GFP and actin from HRT18-∆MUC13 + pMUC13 cells incubated with UV-inactivated W28 bacteria at MOI 50 for 4 h. Conditions also included addition of 1.5 mM sialidase inhibitor (DANA) or 1 × Halt protease and phosphatase inhibitor cocktail (p.i.). Cells were also treated with a combination of 200 U/mL of α2,3,6,8,9 neuraminidase A and 0.6 U of α1,2,3,4,6-L-fucosidase. (H) Immunoblot analysis of MUC13-GFP and actin from HRT18- ∆MUC13 + pMUC13 cells incubated with UV-inactivated W28 bacteria at MOI 120 for 5 h in the absence or presence of 5.5 mM DANA. All immunoblots generated for experiments depicted in (G,H) are available in Figure S5.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Activity Assay, Positive Control, Two Tailed Test, Glycoproteomics, Western Blot, Incubation, Immunoprecipitation, Generated, Bacteria

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 3. Adherence of Bifidobacterium bifidum strains to HRT18 monolayers and penetration of the secreted mucus layer in Caco-2 ALI/VIP cultures. (A) FISH staining in combination with confocal microscopy of UV-killed B. bifidum adhesion to HRT18 monolayers at MOI 10 and 50 for 20 h using a 16S PNA probe (green). White scale bars represent 20 µM. (B) Adhesion of B. bifidum W23, W28, and 20456 strains to HRT18 monolayers assessed by quantification of colony-forming units (CFUs). The graph represents the average and SEM of three independent experiments. Statistical test: one-way ANOVA with Tukey’s correction. (C) Schematic representation of Caco-2 cells cultured in Transwells under liquid–liquid interface (LLI) and air– liquid interface (ALI) with the basolateral addition of vasointestinal peptide (VIP). Caco-2 ALI/VIP cultures produce a secreted mucus layer on the apical surface41 (D) Fluorescence confocal microscopy images of Caco-2 cells grown under ALI/VIP conditions to induce mucus formation incubated with pre-stained W23, W28, 20456, and W1 bacteria (green) at MOI 50 for 4 h. The secreted mucus layer was stained with the lectin Jacalin (orange) and DAPI was used for the nuclei (blue). The middle of the image shows the maximal intensity projection of the mucus and bacterial stainings, while the orthogonal view depicts single planes for clarity. White scale bars represent 20 µM. (E) 3D rendering of images is shown in (B). (F) Immunoblot analysis of MUC13 modification in the Caco-2 ALI/VIP cultures after incubation with B. bifidum strains at MOI 50 for 4 h. All immunoblots used for quantification are available in Figure S6. (G) Quantification of protein abundance of the high and low MW MUC13 (relative to actin) as shown in (D). Graph depicts experimental results of three independent biological replicates. Statistical test: one-way ANOVA with Dunnett’s post hoc test. * p < 0.05.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Staining, Confocal Microscopy, Cell Culture, Fluorescence, Incubation, Bacteria, Western Blot, Modification, Quantitative Proteomics

Journal: Scientific reports

Article Title: Probiotic Bifidobacterium bifidum strains desialylate MUC13 and increase intestinal epithelial barrier function.

doi: 10.1038/s41598-025-92125-2

Figure Lengend Snippet: Fig. 4. Bifidobacterium bifidum strains W23 and W28 enhance intestinal barrier properties. (A) Immunofluorescence confocal images of HRT18 cells grown in glass slides until full confluency and infected with B. bifidum strains W23, W28, and 20456 at MOI 10 for 20 h. Cells were stained for occludin (green), ZO-1 (red), and DAPI (white). The maximum intensity projection is depicted. White scale bars represent 20 µM. (B) Immunofluorescence images of HRT18 cultures grown in Transwells incubated with W23, W28, 20456, W1, and W37 at MOI 100 for 28 h. Cultures were stained for occludin (green), ZO-1 (red), and DAPI (white). Z-stacks were captured from the apical surface until the appearance of nuclei and maximum intensity projections are depicted. White scale bars represent 20 µM. (C) Immunoblot analysis of MUC13 modification and expression of claudins-1, -3, and -4 in HRT18 Transwell cultures after incubation with bacterial strains W23, W28, 20456, W1, and W37 at MOI 100 for 28 h and during a challenge with 100 ng/mL IL-1β and 100 ng/mL TNF-α. The control sample only received the cytokine challenge without bacteria. All immunoblots used for quantification are available in Figure S7. (D) Quantification of protein expression of MUC13 and claudins-1, -3, and -4 (relative to actin) as shown in (C). All graphs depict the SEM of three independent experiments. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (E) TEER measurements of HRT18 Transwell cultures over time during incubation with W23, W28, 20456, W1, and W37 strains at MOI 100 and cytokine challenge at baseline as described for C. (F) TEER values at the 28 h timepoint depicted in E. Graphs depict SEM of three independent biological replicates. Statistical test: two-way ANOVA with Dunnett’s post hoc test. **** p < 0.0001.

Article Snippet: The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS, and infected with bacteria in DMEM medium without FCS at a MOI of 10 and 50 for 20 h at 37 °C in anaerobic Species Former name Strain Medium Reference Lactiplantibacillus plantarum Lactobacillus plantarum W1/ WCFS1 MRS Winclove B.V Lactoccocus lactis subspecies lactis W19 MRS Winclove B.V Lacticaseibacillus paracasei Lactobacillus paracasei W20 MRS Winclove B.V Bifidobacterium bifidum W23 DSMZ #58 Winclove B.V Bifidobacterium bifidum W28 DSMZ #58 Winclove B.V Lactobacillus acidophilus W37 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W51 MRS Winclove B.V Bifidobacterium animalis subspecies lactis W53 MRS Winclove B.V Enterococcus faecium W54 MRS Winclove B.V Lactococcus lactis subspecies lactis W58 MRS Winclove B.V Lacticaseibacillus rhamnosus Lactobacillus rhamnosus W71 MRS Winclove B.V Lactobacillus helveticus W74 MRS Winclove B.V Limosilactobacillus reuteri Lactobacillus reuteri W192 MRS Winclove B.V Lactoccocus lactis subspecies cremoris W224 MRS Winclove B.V Bifidobacterium bifidum DSM 20,456 DSMZ #58 DMSZ Table 1.

Techniques: Immunofluorescence, Infection, Staining, Incubation, Western Blot, Modification, Expressing, Control, Bacteria