Claudin-1 Is a p63 Target Gene with a Crucial Role in Epithelial Development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

ΔΝp63_α functionally interacts <i>in vivo</i> with the <i>Cldn-1</i> gene.

<p>A) Schematic representation of the human, rat and mouse <i>Cldn-1</i> promoter regions. Two regions of high interspecies homology were identified (R1 and R2, black and striped boxes respectively). The degree of homology within R1 and R2 is shown on the right. R3, mapping at −3.5 Kb from the ATG of the <i>Cldn-1</i> gene, did not show any homology and was used as negative control. B) Three amplicons centered on R1, R2 and R3 of the <i>Cldn-1</i> gene were used in semiquantitative PCR amplifications with chromatin extracted from mouse primary keratinocytes, induced with Ca²⁺ for 24 hours, and immunoprecipitated with anti-p63 antibodies (4A4, Santa Cruz Biotech). R1 and R2 were positives with the anti-p63 antibodies while R3 was negative. As positive control, oligonucleotides annealing to the <i>IKKα</i> mouse promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002715#pone.0002715-Marinari1" target="_blank">[25]</a> were used. C) H1299 cells were treated with 20 µM Doxicycline in order to induce ΔNp63α expression. Thirty hours after induction mRNA was extracted from uninduced (-Dox) and induced (+Dox) cells and levels of endogenous <i>Cldn-1</i> and <i>GAPDH</i> were assessed by semiquantitative RT-PCR. D) Expression of the Cldn-1 protein is clearly detected upon ΔNp63α expression in H1299 cells. E) RNA extracted from mouse HL at E10.5, E11.5 and E12.5 (white, grey and black bars respectively) were used to verify <i>ΔNp63</i> and <i>Cldn-1</i> levels of expression.</p

The AEC mutations subvert p63 transcriptional potential.

<p>A) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for TAp63 mutants were cotransfected (0.025, 0.05, 0.1 µg) (striped, light grey and dark grey bars respectively). B) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for ΔNp63 mutants were cotransfected (0.025, 0.05, 0.1 µg) (striped, light grey and dark grey bars respectively). C) 0.05 µg of TAp63α or TA-AEC518 were transfected either alone or together in order to reproduce the heterozygous state found in AEC patients. D) 0.05 µg of ΔNp63α or ΔN-AEC518 were transfected either alone or together in order to reproduce the heterozygous state found in AEC patients. Cells were lysed after 24 hours and luciferase activity was determined. The basal activity of the reporter plasmids was set to 1. Data are presented as fold activation/repression relative to the sample without effectors. Each bar of the histogram represents the mean of three independent transfection duplicates. Standard deviations are indicated.</p

The <i>Cldn-1</i> promoter is regulated by the ΔNp63_ isoforms.

<p>A) Schematic representation of the <i>Cldn-1</i> promoter constructs used. White boxes: <i>Sp1</i> binding sites. Black boxes: <i>p53</i> binding sites. B) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for the p63 isoforms and p53 were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). C) U2OS cells were transfected with the deletion constructs of the <i>Cldn-1</i> reporter plasmid (0.3 µg). Different quantities of expression plasmids for the ΔNp63α were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). D) U2OS cells were transfected with the wild- type or the mutated −61 bp promoter constructs (0.3 µg). Different quantities of expression plasmids for the ΔNp63α were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). Cells were lysed after 24 hours and luciferase activity was determined. The basal activity of the reporter plasmids was set to 1. Data are presented as fold activation/repression relative to the sample without effectors. Each bar of the histogram represents the mean of three independent transfection duplicates. Standard deviations are indicated.</p