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Evaluation of cell surface properties and safety of LAB. (A) Auto-aggregation ability of LAB after 3, 6, and 24 h of static incubation. Statistical significance was assessed relative to strain L2 at 3 and 24 h and relative to strain L5 at 6 h; (B) Cell surface hydrophobicity of LAB assessed using <t>n-hexadecane,</t> ethyl acetate, and xylene, with statistical significance determined relative to strain L2. (C) Adhesion of LAB strains to ileal tissue, with statistical significance determined relative to strain L2. (D) Hemolytic activity of LAB. Data are shown as mean ± SD ( n = 3). Statistical analyses for panels (A–C) were performed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Ns indicates no significant difference; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
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Evaluation of cell surface properties and safety of LAB. (A) Auto-aggregation ability of LAB after 3, 6, and 24 h of static incubation. Statistical significance was assessed relative to strain L2 at 3 and 24 h and relative to strain L5 at 6 h; (B) Cell surface hydrophobicity of LAB assessed using <t>n-hexadecane,</t> ethyl acetate, and xylene, with statistical significance determined relative to strain L2. (C) Adhesion of LAB strains to ileal tissue, with statistical significance determined relative to strain L2. (D) Hemolytic activity of LAB. Data are shown as mean ± SD ( n = 3). Statistical analyses for panels (A–C) were performed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Ns indicates no significant difference; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
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CIT lines express identifiable neoantigens whose cognate CD8+ T cells are responsive to immunotherapy. ( a ) Selection criteria for CIT6 mutations predicted to generate neoantigens. (b) Predicted peptide log affinity ratio (affinity ratio is defined as the ratio between predicted binding affinity of mutant <t>versus</t> <t>wild-type</t> sequence) and gene expression level of mutations predicted to be top CIT6 candidate neoantigens, shown for H2-Dq and H2-Kq alleles. Affinity predictions were generated using the NetH2Pan algorithm. (c) Schematic of IFNγ ELISpot assay. For screening of CD8+ T cell and CD4+ T cell responses, CD8+ or CD4+ T cells were isolated from the spleens of tumor-bearing mice and co-cultured with total splenocytes from a healthy tumor-naïve mouse. (d) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Brinp3 peptide CPAFLPCTV (top row) and negative control (no peptide, bottom row). (e) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Brinp3 peptide CPAFLPCTV. Each colored line represents data points from a different mouse (n = 4 mice). (f) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Ttll4 peptide VPPSSLLPL (top row) and negative control (no peptide, bottom row). (g) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Ttll4 peptide VPPSSLLPL. Each colored line represents data points from a different mouse (n = 4 mice). (h) Spot counts from ELISpot assays performed with CD4+ T cells isolated from the spleens of CIT6-bearing mice and cultured with splenocytes from tumor-naïve mice loaded with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 4 mice). (i) Spot counts from ELISpot assays performed with total splenocytes from CIT6-bearing mice cultured with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 10 – 11 mice). (j) Schematic of ICI treatment regimen and ELISpot analysis. (k) Average spot counts of ICI-treated splenocytes cultured with 80 ug/ml of Ttll4 peptide vs negative control. Each line represents data points from a different mouse (n = 6 mice). Statistical analysis in panels e, g, h, i, and k was done by a paired t- test. ∗ P < .05, ∗∗ P < .01. Error bars in panels e, g, h and i represent mean and SD. CIT, carcinogen-induced tumor; ICI, Immune checkpoint inhibitor.
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CIT lines express identifiable neoantigens whose cognate CD8+ T cells are responsive to immunotherapy. ( a ) Selection criteria for CIT6 mutations predicted to generate neoantigens. (b) Predicted peptide log affinity ratio (affinity ratio is defined as the ratio between predicted binding affinity of mutant <t>versus</t> <t>wild-type</t> sequence) and gene expression level of mutations predicted to be top CIT6 candidate neoantigens, shown for H2-Dq and H2-Kq alleles. Affinity predictions were generated using the NetH2Pan algorithm. (c) Schematic of IFNγ ELISpot assay. For screening of CD8+ T cell and CD4+ T cell responses, CD8+ or CD4+ T cells were isolated from the spleens of tumor-bearing mice and co-cultured with total splenocytes from a healthy tumor-naïve mouse. (d) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Brinp3 peptide CPAFLPCTV (top row) and negative control (no peptide, bottom row). (e) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Brinp3 peptide CPAFLPCTV. Each colored line represents data points from a different mouse (n = 4 mice). (f) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Ttll4 peptide VPPSSLLPL (top row) and negative control (no peptide, bottom row). (g) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Ttll4 peptide VPPSSLLPL. Each colored line represents data points from a different mouse (n = 4 mice). (h) Spot counts from ELISpot assays performed with CD4+ T cells isolated from the spleens of CIT6-bearing mice and cultured with splenocytes from tumor-naïve mice loaded with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 4 mice). (i) Spot counts from ELISpot assays performed with total splenocytes from CIT6-bearing mice cultured with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 10 – 11 mice). (j) Schematic of ICI treatment regimen and ELISpot analysis. (k) Average spot counts of ICI-treated splenocytes cultured with 80 ug/ml of Ttll4 peptide vs negative control. Each line represents data points from a different mouse (n = 6 mice). Statistical analysis in panels e, g, h, i, and k was done by a paired t- test. ∗ P < .05, ∗∗ P < .01. Error bars in panels e, g, h and i represent mean and SD. CIT, carcinogen-induced tumor; ICI, Immune checkpoint inhibitor.
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Evaluation of cell surface properties and safety of LAB. (A) Auto-aggregation ability of LAB after 3, 6, and 24 h of static incubation. Statistical significance was assessed relative to strain L2 at 3 and 24 h and relative to strain L5 at 6 h; (B) Cell surface hydrophobicity of LAB assessed using n-hexadecane, ethyl acetate, and xylene, with statistical significance determined relative to strain L2. (C) Adhesion of LAB strains to ileal tissue, with statistical significance determined relative to strain L2. (D) Hemolytic activity of LAB. Data are shown as mean ± SD ( n = 3). Statistical analyses for panels (A–C) were performed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Ns indicates no significant difference; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Journal: Poultry Science

Article Title: Screening and characterization of Limosilactobacillus reuteri L16 and its probiotic properties in Hy-Line Brown chicks

doi: 10.1016/j.psj.2026.107024

Figure Lengend Snippet: Evaluation of cell surface properties and safety of LAB. (A) Auto-aggregation ability of LAB after 3, 6, and 24 h of static incubation. Statistical significance was assessed relative to strain L2 at 3 and 24 h and relative to strain L5 at 6 h; (B) Cell surface hydrophobicity of LAB assessed using n-hexadecane, ethyl acetate, and xylene, with statistical significance determined relative to strain L2. (C) Adhesion of LAB strains to ileal tissue, with statistical significance determined relative to strain L2. (D) Hemolytic activity of LAB. Data are shown as mean ± SD ( n = 3). Statistical analyses for panels (A–C) were performed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Ns indicates no significant difference; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For the hydrophobicity assay, 3 mL of bacterial suspension was mixed separately with 1 mL of xylene, ethyl acetate (Chronchem, Chengdu, China), or n-hexadecane (Macklin, Shanghai, China).

Techniques: Incubation, Cell Surface Hydrophobicity, Activity Assay

CIT lines express identifiable neoantigens whose cognate CD8+ T cells are responsive to immunotherapy. ( a ) Selection criteria for CIT6 mutations predicted to generate neoantigens. (b) Predicted peptide log affinity ratio (affinity ratio is defined as the ratio between predicted binding affinity of mutant versus wild-type sequence) and gene expression level of mutations predicted to be top CIT6 candidate neoantigens, shown for H2-Dq and H2-Kq alleles. Affinity predictions were generated using the NetH2Pan algorithm. (c) Schematic of IFNγ ELISpot assay. For screening of CD8+ T cell and CD4+ T cell responses, CD8+ or CD4+ T cells were isolated from the spleens of tumor-bearing mice and co-cultured with total splenocytes from a healthy tumor-naïve mouse. (d) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Brinp3 peptide CPAFLPCTV (top row) and negative control (no peptide, bottom row). (e) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Brinp3 peptide CPAFLPCTV. Each colored line represents data points from a different mouse (n = 4 mice). (f) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Ttll4 peptide VPPSSLLPL (top row) and negative control (no peptide, bottom row). (g) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Ttll4 peptide VPPSSLLPL. Each colored line represents data points from a different mouse (n = 4 mice). (h) Spot counts from ELISpot assays performed with CD4+ T cells isolated from the spleens of CIT6-bearing mice and cultured with splenocytes from tumor-naïve mice loaded with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 4 mice). (i) Spot counts from ELISpot assays performed with total splenocytes from CIT6-bearing mice cultured with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 10 – 11 mice). (j) Schematic of ICI treatment regimen and ELISpot analysis. (k) Average spot counts of ICI-treated splenocytes cultured with 80 ug/ml of Ttll4 peptide vs negative control. Each line represents data points from a different mouse (n = 6 mice). Statistical analysis in panels e, g, h, i, and k was done by a paired t- test. ∗ P < .05, ∗∗ P < .01. Error bars in panels e, g, h and i represent mean and SD. CIT, carcinogen-induced tumor; ICI, Immune checkpoint inhibitor.

Journal: JID Innovations

Article Title: CIT tumor lines: A series of immunogenic murine cutaneous squamous cell carcinoma cell lines derived from chemical carcinogenesis

doi: 10.1016/j.xjidi.2026.100477

Figure Lengend Snippet: CIT lines express identifiable neoantigens whose cognate CD8+ T cells are responsive to immunotherapy. ( a ) Selection criteria for CIT6 mutations predicted to generate neoantigens. (b) Predicted peptide log affinity ratio (affinity ratio is defined as the ratio between predicted binding affinity of mutant versus wild-type sequence) and gene expression level of mutations predicted to be top CIT6 candidate neoantigens, shown for H2-Dq and H2-Kq alleles. Affinity predictions were generated using the NetH2Pan algorithm. (c) Schematic of IFNγ ELISpot assay. For screening of CD8+ T cell and CD4+ T cell responses, CD8+ or CD4+ T cells were isolated from the spleens of tumor-bearing mice and co-cultured with total splenocytes from a healthy tumor-naïve mouse. (d) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Brinp3 peptide CPAFLPCTV (top row) and negative control (no peptide, bottom row). (e) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Brinp3 peptide CPAFLPCTV. Each colored line represents data points from a different mouse (n = 4 mice). (f) Representative IFNγ ELISpot images of the assay performed with CD8+ T cells from the spleen of CIT6-bearing mice cultured with 160ug/ml of Ttll4 peptide VPPSSLLPL (top row) and negative control (no peptide, bottom row). (g) Quantification of average IFNγ ELISpot spot counts for CD8+ T cells from the spleen of CIT6-bearing mice cultured with different concentrations of Ttll4 peptide VPPSSLLPL. Each colored line represents data points from a different mouse (n = 4 mice). (h) Spot counts from ELISpot assays performed with CD4+ T cells isolated from the spleens of CIT6-bearing mice and cultured with splenocytes from tumor-naïve mice loaded with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 4 mice). (i) Spot counts from ELISpot assays performed with total splenocytes from CIT6-bearing mice cultured with increasing concentrations of Brinp3 and Ttll4 peptides. Each colored line represents data points from a different mouse (n = 10 – 11 mice). (j) Schematic of ICI treatment regimen and ELISpot analysis. (k) Average spot counts of ICI-treated splenocytes cultured with 80 ug/ml of Ttll4 peptide vs negative control. Each line represents data points from a different mouse (n = 6 mice). Statistical analysis in panels e, g, h, i, and k was done by a paired t- test. ∗ P < .05, ∗∗ P < .01. Error bars in panels e, g, h and i represent mean and SD. CIT, carcinogen-induced tumor; ICI, Immune checkpoint inhibitor.

Article Snippet: Eight-week-old wild-type FVB/N mice were purchased from Jackson Laboratories.

Techniques: Immunopeptidomics, Selection, Binding Assay, Mutagenesis, Sequencing, Gene Expression, Generated, Enzyme-linked Immunospot, Isolation, Cell Culture, Negative Control