Additional file 1: of A review of measures of women’s empowerment and related gender constructs in family planning and maternal health program evaluations in low- and middle-income countries

Evaluation papers included in systematic review. (DOCX 27 kb

Partial Rescue of EDM1 phenotype in skeletal muscle by over-expression of MBNL1.

<p><b>A.</b> RNA-IP using a monoclonal antibody against MBNL1 pulls down the GFP-<i>DMPK</i> 3′UTR mRNA in both the EDM1 and adult-onset DM1 mice but not mice expressing EGFP only. Beads, IgG and RT -ve controls were used to show MBNL1 specificity. EGFP was amplified to detect the transgene. <b>B.</b> MBNL1 was confirmed to be up-regulated by qRT-PCR and <b>C.</b> by western blot analysis in the MBNL1 treated leg but not the EGFP treated leg. <b>D.</b> Median myotonia score in EDM1 mice expressing MBNL1 and EGFP in their TA muscles shows a slight reduction in myotonia due the presence of MBNL1 but not EGFP. <b>E.</b> mRNA splicing was also partially improved for <i>SmyD1</i>. <i>Clcn1</i>-exon7a showed no change. Number of samples (n) provided along with average or median in the tables along with standard deviation. Two Sample T-Test was used to determine statistical significance with p-values displayed.</p

Age of Onset of RNA Toxicity Influences Phenotypic Severity: Evidence from an Inducible Mouse Model of Myotonic Dystrophy (DM1)

<div><p>Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by an expanded (CTG)n tract in the 3′ UTR of the <i>Dystrophia Myotonica Protein Kinase</i> (<i>DMPK</i>) gene. This causes nuclear retention of the mutant mRNA into ribonuclear foci and sequestration of interacting RNA-binding proteins (such as muscleblind-like 1 (MBNL1)). More severe congenital and childhood-onset forms of the disease exist but are less understood than the adult disease, due in part to the lack of adequate animal models. To address this, we utilized transgenic mice over-expressing the <i>DMPK</i> 3′ UTR as part of an inducible RNA transcript to model early-onset myotonic dystrophy. In mice in which transgene expression was induced during embryogenesis, we found that by two weeks after birth, mice reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, muscle weakness, histopathology and mRNA splicing defects. Notably, these defects were more severe than in adult mice induced for an equivalent period of exposure to RNA toxicity. Additionally, the utility of the model was tested by over-expressing MBNL1, a key therapeutic strategy being actively pursued for treating the disease phenotypes associated with DM1. Significantly, increased MBNL1 in skeletal muscle partially corrected myotonia and splicing defects present in these mice, demonstrating the responsiveness of the model to relevant therapeutic interventions. Furthermore, these results also represent the first murine model for early-onset DM1 and provide a tool to investigate the effects of RNA toxicity at various stages of development.</p></div

Lack of parent of origin effect in the EDM1 mice.

<p>Mice born from a sick or healthy dam were not statistically different with regard to grip strength, PR interval or myotonia score at 4 weeks of age.</p

Histopathology, RNA splicing and CUGBP1 are all altered in the EDM1 mice.

<p><b>A.</b> H&E staining shows uniform fibers and peripheral nuclei in the wildtype sections. With RNA toxicity, there is an increase in central nuclei (white arrowhead), increase in fiber size variability, and of atrophic fibers (black arrow). <b>B.</b> CUGBP1 is increased in the skeletal muscles of EDM1 mice. <b>C. </b><i>Clcn1</i>, <i>Nfix</i>, <i>Nrap</i>, <i>Mbnl1</i>, and <i>SmyD1</i> splicing alterations found in human DM1 are present in the EDM1 mice. Number of samples (n) provided in the tables, averages listed in the tables along with standard deviation. Two Sample T-Test was used to determine statistical significance with p-values displayed.</p

Characterization of EDM1 mice.

<p><b>A.</b> Myotonia was measured and scored on a 3 point scale with 0 being no myotonia and 3 being strong, persistent, and prevalent myotonia. EDM1 mice show myotonia as early as 2 weeks of age and reached maximal severity by 4 weeks. <b>B.</b> EDM1 mice have a reduction in grip strength as early as 4 weeks of age while the adult-onset mice did not show a significant reduction. <b>C.</b> PR interval was measured in the EDM1 mice. No statistical difference was measured at 2 weeks of age; however the subsequent time points showed a significant lengthening of the interval. Similar but less severe results were observed in the adult-onset DM1 model. Number of samples (n) provided on the graph, averages listed in the tables below the graphs, error bars are SEM. Two Sample T-Test was used to determine statistical significance with p-values of 0.05 (*), 0.01 (**) or <0.001 (***) displayed.</p

Expression of CELF1 is not affected by absence of PKCα/β.

<p>(<b>A</b>) Western blot analysis of relevant proteins from skeletal muscle extracts of DM5/<i>PKCα/β</i> ^<i>-/-</i> and DM5/<i>PKCα/β</i> ^+/+ uninduced and induced mice at 6 weeks post-induction. Western blot analyses confirmed that PKCα and phospho-PKCα/β were absent in <i>PKCα/β</i> ^-/- mice. Western blot for CELF1 showed 2–4 fold induction in the RNA toxicity mice with or without PKCα/β. There was no difference in MBNL1 expression due to absence of PKCα/β or presence of RNA toxicity. GAPDH or Tubulin was used as a loading control. (<b>B</b>) Quantification of western blots is graphically depicted in right panel. At least 3–5 mice per group were used for analysis. A t-test was used to compare the results from uninduced and induced groups with or without PKCα/β. (*p = 0.05 Student’s t test); error bars are mean±stdev.</p

<i>PKCα/β</i> deficiency does not affect skeletal muscle fiber size distribution in the RNA toxicity mice.

<p>(<b>A</b>) Histogram of skeletal muscle fiber diameter sizing shows that <i>PKCα/β</i> deficiency in the absence of RNA toxicity mice (DM5/<i>PKCαβ</i>^<i>-/-</i> D- (n = 3)) (red) results in a distribution similar to that seen in uninduced mice (DM5/<i>PKCαβ</i>^<i>+/+</i> D- (n = 3)) (blue). (<b>B</b>) Histogram of skeletal muscle fiber diameter distribution shows that smaller fibers in the RNA toxicity mice (DM5 D+ (n = 3)) (red) as compared to uninduced mice (DM5 D- (n = 3)) (blue). (<b>C</b>) Histogram of skeletal muscle fiber diameter sizing shows that no differences in fiber size distribution after <i>PKCα/β</i> deficiency (blue) in the presence of RNA toxicity (red). (<b>D</b>) The graph showing no differences in the percentage of fibers less than 50μm in the <i>PKCα/β</i> deficiency mice as compared to normal mice in the presence of RNA toxicity. Note the increased percentage fibers in the presence of toxic RNA as compared to uninduced mice. At least 300 fibers were analyzed per mouse. *p = 0.05, and ***p = 0.001, Student’s t test; error bars are mean±stdev.</p

Muscle histopathology in the RNA toxicity mice is not corrected by absence of PKCα/β.

<p>(<b>A</b>) Hematoxylin and eosin (H&E) staining was performed on quadriceps muscles form DM5/<i>PKCα/β</i> ^<i>-/-</i> and DM5/<i>PKCα/β</i> ^+/+ mice in the absence or presence of RNA toxicity. A representative H&E section is shown for each group. Uninduced DM5/<i>PKCα/β</i> ^<i>-/-</i> and DM5/<i>PKCα/β</i> ^+/+ mice showed normal muscle histology. By 6 weeks of 0.02% doxycycline induction, we saw similar level of histopathology in the presence or absence of PKCα/β. (<b>B</b>) In the RNA toxicity mice; ~20% of nuclei were centralized. No significant difference was observed in the percentage of centrally nuclei between DM5/<i>PKCα/β</i> ^<i>-/-</i> D+ and DM5/<i>PKCα/β</i> ^+/+ D+ mice. For each groups, at least 4–5 mice were analyzed. At least 300 fibers were analyzed per mouse. ***p = 0.001, Student’s t test; error bars are mean±stdev.</p

Phospho-PKCθ levels are increased in the RNA toxicity mice.

<p>(<b>A, B</b>) Western blot and quantitative RT-PCR shows no significant differences in PKCθ expression in uninduced and induced skeletal muscle tissues from RNA toxicity mice (n = 3–5 for uninduced and n = 3–5 for induced mice). Tubulin-loading control. (<b>C</b>) Western blot of skeletal muscle protein extracts shows increased levels of p-PKCθ in DM5 mice with RNA toxicity. n.s. indicates non-specific band; GAPDH-loading control. *p = 0.05, Student’s t test; error bars are mean±stdev.</p