Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: EMBO Reports

Article Title: Small‐molecule inhibition of aging‐associated chromosomal instability delays cellular senescence

doi: 10.15252/embr.201949248

Figure Lengend Snippet: A Mitotic duration of n = 100 neonatal (N) and elderly (87 years) human dermal fibroblasts (HDF) treated with DMSO (−) or UMK57 (+) for 96 h. B Relative calcium‐stable k‐fiber intensity levels scored by immunofluorescence analysis of n ≥ 29 tubulin‐stained metaphase cells of neonatal and elderly samples treated with DMSO and UMK57 for 96 h. Levels were normalized to neonatal DMSO‐treated condition. C Aneusomy index of chromosomes 7, 12, and 18 measured by interphase FISH analysis of neonatal and elderly n = cells treated for 96 h. D Cytochalasin D‐induced binucleated (BN) n = cells with mis‐segregation (MS) of chromosomes 7, 12, and 18 in neonatal and elderly samples treated for 96 h. E Percentage of n = cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥ 1 foci; DNA damage) senescence biomarkers after 96 h of treatment. F Percentage of n = cells staining positive for SA‐β‐gal activity when treated for 96 h with DMSO or UMK57. G, H Representative images (G) and quantification (H) of phospho‐Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n ≥ 75 neonatal and elderly metaphase cells treated with UMK57 for 24 or 96 h. Intensity levels were normalized to ACA. Scale bar, 5 μm. Data information: All values are mean ± SD of at least two independent experiments. ns P > 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001 by two‐tailed (A, B, H) Mann–Whitney and (C–F) chi‐square tests.

Article Snippet: Primary antibodies: rabbit anti‐53BP1 (4937, Cell Signaling Technology), 1:100; mouse anti‐p21 (SC‐6246, Santa Cruz Biotechnology), 1:800; mouse anti‐Aurora B (Aim‐1; 611082, BD Biosciences), 1:500; rabbit anti‐cGAS (15102, Cell Signaling Technology), 1:200; mouse anti‐Hec1 (ab3613, Abcam), 1:1,500; mouse anti‐Plk1 (SC‐17783, Santa Cruz Biotechnology), 1:2,000; rabbit anti‐MCAK , 1:5,000; mouse anti‐α‐Tubulin (T5168, Sigma‐Aldrich), 1:1,500; human anti‐centromere antibody (ACA; kindly provided by Dr. W. C. Earnshaw), 1:3,000; rabbit anti‐Aurora B phospho T232 (600‐401‐677, ROCKLAND), 1:1,000; mouse anti‐Retinoblastoma (554136, BD Biosciences), 1:100.

Techniques: Immunofluorescence, Staining, Double Immunostaining, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Small‐molecule inhibition of aging‐associated chromosomal instability delays cellular senescence

doi: 10.15252/embr.201949248

Figure Lengend Snippet: A Percentage of micronuclei in n = cells of neonatal versus elderly samples after 96‐h treatment. B Percentage of cGAS + /Rb − and cGAS − /Rb + micronuclei in n = cells scored by immunofluorescence analysis upon 96‐h treatment. C Relative CDKN1A / P21, FAM214B, CCND1, B4GALT7, and TOLLIP transcript levels in total RNA of neonatal (HDF N; n = 5 replicates) and elderly (HDF 87 years; n = 3 replicates) fibroblasts treated with DMSO or UMK for 96 h. TBP and HPRT1 were used as reference genes. All levels were normalized to DMSO‐treated neonatal sample. D Experimental layout for prolonged exposure to UMK57 of neonatal (N) and elderly (87 years) fibroblast cultures, with cell passage every week and media renewal halfway each week. At week 4, chromosome segregation and senescence biomarkers were analyzed. E Aneusomy index of chromosomes 7, 12, and 18 measured by interphase FISH in n = cells. F Percentage of n = cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥ 1 foci; DNA damage) senescence biomarkers. G Percentage of n = cells staining positive for SA‐β‐gal activity. H Calcium‐stable k‐fiber intensity levels scored by immunofluorescence analysis of n ≥ 33 tubulin‐stained mitotic cells in prometaphase of neonatal and elderly samples treated with DMSO (−) and UMK57 (+) for 96 h. Levels were normalized to neonatal DMSO‐treated condition. I Phospho‐Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres of neonatal and elderly prometaphase cells treated with DMSO (−) or UMK57 (+) for 24 or 96 h. Intensity levels were normalized to ACA. Data Information: All values are mean ± SD of at least two independent experiments. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two‐tailed (A, B, E–G) chi‐square and (C, H, I) Mann–Whitney tests.

Article Snippet: Primary antibodies: rabbit anti‐53BP1 (4937, Cell Signaling Technology), 1:100; mouse anti‐p21 (SC‐6246, Santa Cruz Biotechnology), 1:800; mouse anti‐Aurora B (Aim‐1; 611082, BD Biosciences), 1:500; rabbit anti‐cGAS (15102, Cell Signaling Technology), 1:200; mouse anti‐Hec1 (ab3613, Abcam), 1:1,500; mouse anti‐Plk1 (SC‐17783, Santa Cruz Biotechnology), 1:2,000; rabbit anti‐MCAK , 1:5,000; mouse anti‐α‐Tubulin (T5168, Sigma‐Aldrich), 1:1,500; human anti‐centromere antibody (ACA; kindly provided by Dr. W. C. Earnshaw), 1:3,000; rabbit anti‐Aurora B phospho T232 (600‐401‐677, ROCKLAND), 1:1,000; mouse anti‐Retinoblastoma (554136, BD Biosciences), 1:100.

Techniques: Immunofluorescence, Staining, Double Immunostaining, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: Serum proteins separated by two dimensional gel electrophoresis in early and advanced NAFLD, with and without HCC . These are representative blots comparing immune depleted and desalted serum from a patient with NAFLD and no fibrosis, a patient with NAFLD cirrhosis, and a patient with NAFLD cirrhosis and cancer. Serum profiles separated by 2D gel electrophoresis from 5 patients in each group, run in duplicate, were compared using Progenesis software. 5 spots were characterised by MS after excision. Spots 1,2 and 3 were isoforms of apolipoprotein A1, spot 4 was apolipoprotein A4, and spot 5 was identified as CD5 antigen like, or CD5L.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Software

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates poorly between cirrhotic patients with and without HCC . Serum levels of CD5L were measured by ELISA assay in cirrhotic patients with (218 ± 221; n = 45) and without (194 ± 166; n = 49) HCC, as shown in 2A. Characteristics of the individuals are shown in Table 1. There was no significant difference between the two groups. ROC analyses is shown in 2B. The area under the curve is 0.495 (95% confidence intervals 0.376 and 0.614). A level > 400 ng/ml has a sensitivity of only 20%, but a specificity of 88%. Levels > 500 ng/ml have a specificity of 96%, > 600 ng/ml of 98% and > 700 ng/ml of 100%. For comparison, the mean CD5L serum level from patients without cirrhosis (detailed in figure 3) is also shown.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates between steatohepatitis patients with and without cirrhosis . Serum levels of CD5L were measured in a total of 113 patients with either ALD or NAFLD. Fibrosis was scored histologically on liver biopsy as per the Brunt scoring system. The numbers of individuals in each group are shown within the boxes of chart 3A. The difference in CD5L levels between the different stages of fibrosis is statistically significant by univariate analysis (p = 0.004) controlled for both age and sex. This difference The ROC analyses for the identification of those individuals with stage 4 fibrosis (cirrhosis) is depicted in 3B. The area under the curve is 0.719 (95% confidence intervals 0.623 and 0.816), p < 0.0001. A level of CD5L 50 ng/ml has a sensitivity of 78% and a specificity of 46%, while a level of 100 ng/ml has a sensitivity of 63% and a specificity of 72%. A level greater than 200 ng/ml has a specificity of 95% (sensitivity 41%), and greater than 300 ng/ml of 97% (sensitivity 27%).

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques:

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L mRNA expression is not altered in association with either fat, inflammation or fibrosis scores in pre-cirrhotic NAFLD liver tissues . mRNA CD5L expression was quantified by real-time PCR, relative to GAPDH and a normal liver sample, in 21 pre-cirrhotic NAFLD biopsy tissues, 13 normal liver samples taken at the time of liver resection, one cirrhotic liver and one HCC. As shown in 4A-C, there was no difference in any pre-cirrhotic NAFLD biopsy tissues in association with the degree of fat, inflammation or fibrosis. There was a significant increase in the NAFLD tissues as a group (n = 21), compared with normal liver tissues (n = 13) as represented in 4D (6.945 ± 0.722 versus1.68 ± 0.269; p = 0.000, ***). The elevated CD5L mRNA expression in one cirrhotic and HCC tissue pair, obtained at the time of laparoscopic radiofrequency ablation, is also presented in 4C and is in keeping with the elevated serum CD5L levels identified in the larger cohort of patients studied.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: The identification of the differentiating protein spots on 2D gels.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Sequencing