Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Redox Biology

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1

doi: 10.1016/j.redox.2019.101321

Figure Lengend Snippet: Multi-omic analysis of the response of keratinocytes to CBD. The transcriptomic and proteomic profiling of RNA and protein samples was carried out using RNA-Seq and LC-MS/MS, respectively. (A, B) Volcano plots showing the magnitude (log2 fold change) and significance (-log10 p value) of the changes in the transcriptomic and proteomic comparisons of CBD treated keratinocytes versus controls (n = 3 for RNA-Seq and n = 4 for proteomics). Every point represents a gene/protein and the colour indicates those surpassing the cut-off of an adjusted P value < 0.05 and an absolute fold change >2 (for genes) or > 1.5 (for proteins). For RNA-Seq, a small value (1e-300) was added to p values in order to avoid logarithms of zero at plotting. (C) Upset plot indicating the overlap between the sets of up or down regulated genes and proteins as a bar plot over a coincidence histogram. (D) Over-representation analysis results. The dot plot indicates with a point the significant over-representation of a given term, transcription factor or pathway in a group of up or down regulated genes/proteins (Fisher Exact Test adjusted P < 0.1). While the colour indicates the adjusted P value of the enrichment, the size of the point represents the enrichR combined score.

Article Snippet: Normal human epidermal keratinocytes (NHEK) and Keratinocyte growth medium were purchased to Innoprot SL (Biscaia, Spain).

Techniques: RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Redox Biology

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1

doi: 10.1016/j.redox.2019.101321

Figure Lengend Snippet: Validation of the NRF2 pathway as a target of CBD in keratinocytes. (A) Gene set enrichment analysis plot for the NRF2 signalling pathway transcriptomic changes. Black lines indicate the position of NRF2 genes in the pre-ranked gene list and the green line indicates the running enrichment score. (B) Magnitude of the changes for significantly up-regulated genes and proteins selected using the previously mentioned cut-offs in the CBD versus control comparison . C) Primary human keratinocytes were incubated with either DMSO or CBD (10 μM) for 24 h. The mRNA levels for HMOX1 ( upper panel ) and SQSTM1 ( p62) ( lower panel ) were quantified using real-time PCR. The data were normalized using HPRT1 as an internal control. Data represent means ± SD (n = 3) and are expressed relative to the DMSO sample. ***P ≤ 0.001 . D) HaCaT cells were incubated with either DMSO or increasing concentration of CBD for 16 h. The mRNA levels for HMOX1 ( upper panel ) and SQSTM1 ( p62) ( lower panel ) were quantified using real-time PCR as previously indicated (n = 3) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. E) HaCaT-ARE-Luc cells were treated with either SFN or CBD at the indicated concentrations for 6 h. Luciferase activity was measured in the cell lysates and expressed as RLU (x 10 4 ). Data represent means ± SD (n = 4) and are expressed relative to untreated cells. **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Normal human epidermal keratinocytes (NHEK) and Keratinocyte growth medium were purchased to Innoprot SL (Biscaia, Spain).

Techniques: Incubation, Real-time Polymerase Chain Reaction, Concentration Assay, Luciferase, Activity Assay

Journal: Redox Biology

Article Title: Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1

doi: 10.1016/j.redox.2019.101321

Figure Lengend Snippet: CBD treatment increases the keratinocyte layer in the epidermis and the expression of HMOX1. (A) Haematoxylin-eosin staining of 5 μm paraffin-embedded sections were analysed by bright field microscopy. (B) Representative images of HMOX1 immunohistochemistry from mouse skin after 5 days of treatment with vehicle, CBD 0.1% or 1% (C) Quantification of HO-1 stained area in mouse epidermis. *P < 0.05, ***P < 0.001 compared with control. Scale bars: 200 (left) and 100 μm (right). n = 15 for vehicle treated samples and n = 12 for CBD treated samples.

Article Snippet: Normal human epidermal keratinocytes (NHEK) and Keratinocyte growth medium were purchased to Innoprot SL (Biscaia, Spain).

Techniques: Expressing, Staining, Microscopy, Immunohistochemistry