Effects of Hydrogen Peroxide on Different Models of Wound Healing

Ph.DDOCTOR OF PHILOSOPH

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

10.1371/journal.pone.0049215PLoS ONE711

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Wounding increases ERK1/2 and p38 phosphorylation which can be further increased by 166 mM H₂O₂ treatment.

<p>(A) Representative blots of wound tissues lysate collected 30 min after wounding. Skin denotes skin from non-wounded animals while control refers to wounds treated with PBS. (B) The density of phosphorylated ERK and pan-ERK and (C) phosphorylated p38 and pan p38 were normalized against α-tubulin re-probed from their respective blot. Results shown are mean ± S.E.M. (n = 4). Densitometry results were analyzed by 1-way ANOVA and test of significance between all column was determined using Tukey’s post hoc test. Only the comparison between 166 mM treated wounds and skin was statistically significant for both B and C. ** p<0.01.</p

Wounding increased lipid peroxidation and nitrative damage but not protein carbonylation.

<p>Levels of F₂-isoprostanes levels in skin and wounds were compared by normalizing against arachidonic acid (A) or tissue weight (B). Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H₂O₂ wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (C) Levels of arachidonic acid in skin and wound tissues. Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H₂O₂ wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (D) Levels of protein carbonyls in wounds were compared to intact skin and expressed as fold change. The results shown are the mean fold change ± S.E.M. No difference in the levels of protein carbonyl was observed in control wounds and 166 mM H₂O₂ treated wounds. (E) Comparison of 3-nitrotyrosine level in skin and wounds. Results shown are mean ± S.E.M., n = 5. The 3-nitrotyrosine levels of skin were compared to control wounds or H₂O₂ treated wounds and analyzed with 1-way ANOVA followed by Dunnett’s post-hoc test. Levels of 3-nitrotyrosine were significantly higher at day 6 after wounding. Levels of 3-nitrotyrosine in control and 166 mM H₂O₂ treated wounds were also compared using 2-way ANOVA and the differences were not statistically significant. *p<0.05, ** p<0.01, ***p<0.001.</p

Low concentrations of H₂O₂ increased wound angiogenesis.

<p>Paraffin sections from day 6 wounds were stained for CD31 using an immunohistochemical method. Representative photomicrograph of (A) control, (B) 166 mM H₂O₂ and (C) 10 mM H₂O₂ treated wounds are shown. (D) The number of brown lumen-like structures within the neodermis was counted in a single blinded fashion and analyzed using 1-way ANOVA followed by Dunnett’s multiple comparison test with control. Graph shown is the mean ± S.E.M, n = 6–7, *** p<0.001.</p

High concentrations of H₂O₂ increase levels of MMP-8 in wounds.

<p>Western blot analysis of wound tissues lysate collected 6 days after wounding. Each lane represents a sample from a different animal. (A) Representative blot of MMP-8. (B) Densitometry analysis of MMP-8 normalized against α-tubulin re-probed from their respective blot. Results are mean ± S.E.M. (n = 4) and were analyzed using 1-way ANOVA followed by Tukey multiple comparison among all the columns. ** p<0.01 (C) Representative blot of MMP-9. (D) Densitometry analysis of MMP-9 normalized against α-tubulin re-probed from their respective blot. Results are mean ± S.E.M. (n = 4), p values for 1-way ANOVA is p = 0.13.</p

High concentrations of H₂O₂ retard connective tissue formation.

<p>Paraffin sections from day 6 wounds were stained with the Masson-Goldner trichrome stain as described in material and method. Connective tissues are stained green. Fibrin, eschar and cytoplasm are stained red. Nuclei are stained dark brown. Representative images for control (A,D) 10 mM (B, E) and 166 mM (C, F) treated wounds are shown. Images A-C are at 100X magnification while D-F are at 200X magnification. (G) Quantification of the fraction of pixels that are stained green. The number of pixels stained green within the neodermis at 100X magnification was quantified using a custom software. The area quantified is outlined with the dashed line. Results were analyzed using 1-way ANOVA followed by Dunnett’s multiple comparison test with control. Graph shown is the mean ± S.E.M. n = 6–7, *** p<0.001.</p

Sex differences in oncogenic mutational processes

Funder: Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology); doi: https://doi.org/10.13039/501100002790Funder: Genome Canada (Génome Canada); doi: https://doi.org/10.13039/100008762Funder: Canada Foundation for Innovation (Fondation canadienne pour l'innovation); doi: https://doi.org/10.13039/501100000196Funder: Terry Fox Research Institute (Institut de Recherche Terry Fox); doi: https://doi.org/10.13039/501100004376Abstract: Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts