Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Long-Term Efficacy of AAV9-U7snRNA-Mediated Exon 51 Skipping in mdx52 Mice

doi: 10.1016/j.omtm.2020.04.025

Figure Lengend Snippet: AAV9-Ex51 Induces Long-Term Functional Improvement (A) Fall latency in the inverted grid test in AAV9-Ex51-treated mice (n = 3), WT mice (n = 4), and control PBS mdx52 mice (n = 5). A maximum score of 120 s was given when the mice did not fall. (B) Fall latency in the wire suspension test in AAV9-Ex51-treated mice (n = 3), WT mice (n = 4), and control PBS mdx52 mice (n = 5). (C) Levels of MYOM3 in serum 8 weeks (n = 3) or 6 months (n = 3) after AAV9-ex51 injection as quantified by western blot using Empiria Studio (LI-COR Biosciences) and normalized to control mdx52 mice (n = 6). (D) Serum CK, urea, creatinine, albumin, AST, ALP, ALT, and bilirubin levels were measured 8 weeks (n = 3) or 6 months (n = 3) after AAV9-ex51 injection compared to WT (n = 11) and control mdx52 mice (n = 9). Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (Mann-Whitney U tests).

Article Snippet: MYOM3 protein was detected by probing the nitrocellulose membrane with MYOM3 primary rabbit polyclonal antibody (Proteintech, Manchester, UK), followed by incubation with a goat anti-rabbit secondary antibody (IRDye 800CW goat anti-rabbit immunoglobulin G [IgG], LI-COR Biosciences, Germany).

Techniques: Functional Assay, Control, Suspension, Injection, Western Blot, MANN-WHITNEY

Journal: Wellcome Open Research

Article Title: Longitudinal assessment of blood-borne musculoskeletal disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.12688/wellcomeopenres.17398.2

Figure Lengend Snippet: Figure a ) representative MYOM3 western blot. This western blot shows a positive control mini-dilution series that was run alongside samples in every gel, followed by 5 samples of WT control dog serum, 5 samples of DE50-MD dog serum, and a negative control lane in which no protein was loaded. Figure b ) MYOM3 quantified by western blot in serum samples from dogs studied longitudinally between 1 and 18 months of age. DE50-MD: grey, total N=83 samples, from N=10 dogs, N=2-8 per age group; WT dogs: (not shown, see Underlying data ), total N=113 samples, from N=10 dogs, N=3-9 per age group. Bands corresponding to MYOM3 were not detected in any WT serum samples tested. Boxes extend from the 25 th to 75 th percentile, with a line within the box at the median value. Each point represents an individual sample, and whiskers show the minimum and maximum results for that age-group. Band intensity calculated by densitometry using ImageJ software. Figure c ) correlation between MYOM3 quantity and CK activity in blood samples from DE50-MD dogs studied longitudinally between 1 and 18 months, total of N=75 samples, from N=10 dogs. (MYOM3: myomesin-3; WT: wild type.)

Article Snippet: Membranes were blocked in 10% milk powder (Marvel) in phosphate buffered saline (PBS, Fisher Scientific, #BR0014G)/Tween 0.05% (Sigma, #P9416) for one hour at room temperature, before incubation with a polyclonal rabbit anti-human MYOM3 antibody (Proteintech, #17692-1-AP, 1:1000 dilution) and a polyclonal rabbit anti-canine albumin antibody (as internal loading control, Biorbyt Ltd, #orb242465; 1:1,000,000 dilution) overnight at 4 °C.

Techniques: Western Blot, Positive Control, Control, Negative Control, Software, Activity Assay

Journal: Wellcome Open Research

Article Title: Longitudinal assessment of blood-borne musculoskeletal disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.12688/wellcomeopenres.17398.2

Figure Lengend Snippet: Included in the analysis was plasma CK activity (log10 U/l), serum MYOM3 quantity (AU), serum MSTN concentration (ng/ml), serum miR-1, miR-133a and miR 206 levels (log10 relative quantity). Study population consisted of DE50-MD and wild-type (WT) dogs studied longitudinally between 3 and 18 months of age. DE50-MD: grey, N=10 dogs total, N=2-9 per age group; WT: white, N=7 dogs total, N=2-5 per age group. Where an individual was missing data for a specific biomarker, missing data was replaced with the mean of the relevant genotype and age-group for that biomarker. Boxes extend from the 25 th to 75 th percentile, with a line within the box at the median value. Each point represents an individual sample, and whiskers show the minimum and maximum results for that age-group. Asterisks denote the level of significance of a difference between genotypes based on linear mixed model analysis: ****P<0.0001. Letters a, b and c denote statistically significant differences (P<0.05) in the mean within either the DE50-MD (grey letters) or WT (black letters) genotypes: means sharing a letter are not significantly different within each genotype group. (CK: creatine kinase; WT: wild type.)

Article Snippet: Membranes were blocked in 10% milk powder (Marvel) in phosphate buffered saline (PBS, Fisher Scientific, #BR0014G)/Tween 0.05% (Sigma, #P9416) for one hour at room temperature, before incubation with a polyclonal rabbit anti-human MYOM3 antibody (Proteintech, #17692-1-AP, 1:1000 dilution) and a polyclonal rabbit anti-canine albumin antibody (as internal loading control, Biorbyt Ltd, #orb242465; 1:1,000,000 dilution) overnight at 4 °C.

Techniques: Clinical Proteomics, Activity Assay, Concentration Assay, Biomarker Discovery

Journal: Wellcome Open Research

Article Title: Longitudinal assessment of blood-borne musculoskeletal disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.12688/wellcomeopenres.17398.2

Figure Lengend Snippet: Sample size calculations. N required to show with sufficient power (0.8) an improvement in a DE50-MD biomarker towards WT levels with any given treatment. Sample sizes were calculated for the principal component analysis output (performed on blood-borne CK activity, MYOM3 quantity, MSTN concentration, and miR-1, -133a and -206 relative quantity) and for each individual biomarker that was significantly elevated in DE50-MD compared to WT blood samples for dogs aged 3–18 months. (CK: creatine kinase; MSTN: myostatin; MYOM3: myomesin-3; WT: wild type.)

Article Snippet: Membranes were blocked in 10% milk powder (Marvel) in phosphate buffered saline (PBS, Fisher Scientific, #BR0014G)/Tween 0.05% (Sigma, #P9416) for one hour at room temperature, before incubation with a polyclonal rabbit anti-human MYOM3 antibody (Proteintech, #17692-1-AP, 1:1000 dilution) and a polyclonal rabbit anti-canine albumin antibody (as internal loading control, Biorbyt Ltd, #orb242465; 1:1,000,000 dilution) overnight at 4 °C.

Techniques: Biomarker Discovery, Activity Assay, Concentration Assay