Journal: bioRxiv

Article Title: Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds

doi: 10.1101/2023.06.30.547263

Figure Lengend Snippet: ( A ) Experimental overview for RNA sequencing performed on diabetic murine wounds treated with A. faecalis (AF), S. aureus (SA), or vehicle control (PBS); n = 4 mice/group; 1 wound/mouse. ( B ) Principal component analysis on day 3 wounds demonstrates distinct clustering of the three treatment groups. ( C ) Heatmap of differential expressed genes (DEG with FDR < 0.01) between the AF and PBS groups at day 3. Unsupervised clustering using Spearman’s correlation was used for hierarchical clustering of the samples, while Pearson’s correlation was used for gene clustering (heatmap rows, dendrogram not shown). Colors are used to codify log fold change values, with red values for increased expression and blue values for decreased expression. ( D ) Volcano plot demonstrating differentially expressed genes in AF vs. PBS wounds at day 3. Horizontal dashed line represents a cut off for the FDR p-adjusted value of <0.1. Vertical lines denote a fold change of ±1.5-fold. Genes that surpass both the p-value and fold change cut off are marked in red, with genes that are downregulated by AF to the left of the plot and upregulated to the right. The inset shows the same volcano plot but at higher resolution. Significant genes of interest are highlighted with gene names on the plot. ( E ) Gene ontology (GO) enrichment was performed on differentially expressed genes for AF vs PBS at day 3. Up and downregulated GO terms of interest are highlighted, with the number of genes matching the term adjacent to the bar plot. ( F ) Gene expression changes of MMPs and related genes in AF vs PBS and SA vs PBS day 3 wounds; * p adj <0.05; **** p adj <0.5 × 10 −4 . ( G ) Conceptual model demonstrating the activity of MMP-10 at the epidermal wound tongue.

Article Snippet: For addition of exogenous MMP-10 to the scratches, murine recombinant MMP-10 (RayBiotech 230-00748-10) was diluted in keratinocyte media to the indicated concentrations.

Techniques: RNA Sequencing Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds

doi: 10.1101/2023.06.30.547263

Figure Lengend Snippet: ( A ) Immunofluorescence staining was performed on paraffin-embedded sagittal sections of day 3 wound tongues. Keratin-14 (K14), MMP-10, and nuclear (DAPI) co-staining were performed. Scale bar = 1000 μm. Blue and green boxes indicate locations of higher magnification insets to show greater detail in ( B ). The positive MMP-10 signal is highlighted by arrowheads in the proximal wound edge, while there is negative MMP-10 staining in the distal wound epidermis. Scale bar = 100 μm. ( C ) The percentage of epidermal area with a positive MMP-10 signal was calculated in the epidermis adjacent to the wound bed. The green dashed line highlights the proximal wound tongue, while the blue dashed line is distal wound epithelium. Arrowheads highlight areas of MMP-10 positive signal. Scale bar = 250 μm, n = 4 mice/group; p = 0.081 by Wilcoxon-rank sum test. ( D ) Gaps were created in diabetic mouse keratinocytes, and in vitro wound closure was measured at 24 hours. Initial gap size is drawn in black, and migrating margins are drawn in yellow. AF increases scratch closure, while addition of 200 ng/mL murine recombinant MMP-10 (rMMP-10) abrogates the pro-healing phenotype. [n = 5 independent gaps/group (except n=6 for AF condition); data shown is representative of >3 independent experiments; **: p <= 0.01 by Wilcoxon rank-sum test]. ( E ) 8mm wounds were created in diabetic mice and colonized with PBS, AF, or AF + rMMP-10. Wound images were taken at days 0 and 3 and wound size compared to original wound area was quantified, demonstrating a significant decrease in AF-treated wound size at day 3. Addition of rMMP-10 abolishes AF-mediated accelerated closure. Scale bars = 1 cm; n = 5 mice per group (4 per group for AF + rMMP), and 2 wounds per mouse; *: p <= 0.05; **: p <= 0.01 by Wilcoxon rank-sum test.

Article Snippet: For addition of exogenous MMP-10 to the scratches, murine recombinant MMP-10 (RayBiotech 230-00748-10) was diluted in keratinocyte media to the indicated concentrations.

Techniques: Immunofluorescence, Staining, In Vitro, Recombinant

Journal: bioRxiv

Article Title: Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds

doi: 10.1101/2023.06.30.547263

Figure Lengend Snippet: ( A ) Sample representation of MMP-10 quantification using ImageJ. Shown is a wound image with thresholding performed so that positive pixels are red. The epidermal selection where quantification was performed is outlined in yellow. ( B ) Bacterial CFU quantification of day 3 wounds shown in . On the right, representative blood agar plates from wound bacterial cultures are shown. The AF group is predominated by grey A. faecalis colonies. In the AF + rMMP-10 group, blue arrows point to examples of A. faecalis isolates; white colonies are the murine skin commensal Staphylococcus xylosus (colony identities confirmed by 16s rRNA gene sequencing). n of 5 mice per group for AF, 4 per group for AF + rMMP-10 and 1 wound per mouse. ( C ) Masson’s Trichrome staining of wound sections shown in . The epidermis is stained in purple, and underlying collagen-rich dermis is blue, allowing for clear visualization of re-epithelialization at the wound tongue (i, ii). Representative images of each treatment group are shown. Scale bar = 250 μm.

Article Snippet: For addition of exogenous MMP-10 to the scratches, murine recombinant MMP-10 (RayBiotech 230-00748-10) was diluted in keratinocyte media to the indicated concentrations.

Techniques: Selection, Sequencing, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Mixture of Extracts of Kochia scoparia and Rosa multiflora with PPAR α/γ Dual Agonistic Effects Prevents Photoaging in Hairless Mice

doi: 10.3390/ijms17111919

Figure Lengend Snippet: Kochia scoparia (Ks) and Rosa multiflora (Rm) inhibit UVB-induced MMP-1 expression. Normal human fibroblast (NHF) cells were treated with the methylene chloride (MC) fraction of Ks or Rm (10 µg/mL for each) for 1 h, exposed to UVB irradiation (20 mJ/cm 2 ) and treated with 10 µg/mL of the MC fraction of Ks or Rm for 48 h to measure MMP-1 expression. Relative MMP-1 protein expression in Ks- or Rm-treated NHF cells was determined by ELISA. Ks and Rm significantly inhibited MMP-1 secretion in UVB-irradiated NHF cells. Con: sham light control, UV: UVB irradiation, Ks: Kochia scoparia treatment, Rm: Rosa multiflora treatment. Each bar represents the mean ± SEM of duplicates. * p < 0.05 vs. control; # p < 0.05 vs. UV.

Article Snippet: After UVB irradiation, cells were incubated with the MC fraction of Ks (10 µg/mL) or Rm (10 µg/mL) for another 48 h. MMP-1 secretion was quantified from supernatants using a human MMP-1 ELISA Kit (Merck & Co., Inc., Whitehouse Station, NJ, USA).

Techniques: Expressing, Irradiation, Enzyme-linked Immunosorbent Assay