The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

Bone cells constitutively release ATP into the extracellular environment where it acts locally via P2 receptors to regulate bone cell function. Whilst P2Y receptor stimulation regulates bone mineralisation, the functional effects of this receptor in osteoclasts remain unknown. This investigation used the P2Y receptor knockout ( ) mouse model to investigate the role of this receptor in bone. MicroCT analysis of mice demonstrated age-related increases in trabecular bone volume (≤48%), number (≤30%) and thickness (≤17%). osteoblasts displayed a 3-fold increase in bone formation and alkaline phosphatase activity, whilst osteoclasts exhibited a 65% reduction in resorptive activity. Serum cross-linked C-telopeptide levels (CTX, resorption marker) were also decreased (≤35%). The resorption defect in osteoclasts was rescued by the addition of exogenous ATP, suggesting that an ATP deficit could be a key factor in the reduced function of these cells. In agreement, we found that basal ATP release was reduced up to 53% in osteoclasts. The P2Y receptor agonists, UTP and 2-thioUTP, increased osteoclast activity and ATP release in wild-type but not in cells. This indicates that the P2Y receptor may regulate osteoclast function indirectly by promoting ATP release. UTP and 2-thioUTP also stimulate ATP release from osteoblasts suggesting that the P2Y receptor exerts a similar function in these cells. Taken together, our findings are consistent with the notion that the primary action of P2Y receptor signalling in bone is to regulate extracellular ATP levels.

Background During the late stage of keratinocyte differentiation, corneocytes gain a strong protein–lipid structure: the corneocyte envelopes (CE), composed of the inner corneocyte protein envelope (CPE) and the outer corneocyte lipid envelope (CLE). The hydrophobicity of CEs depends on the covalent attachment of linoleoyl‐acyl‐ceramides by transglutaminases (TG). These ceramides are processed by a range of other enzymes, including 12R‐lipoxygenase (12R‐LOX), before the covalent attachment of the free ω‐hydroxyceramides to the CPE surface to form the CLE. The mechanical strength of CE is obtained with the formation of isodipeptide bonds by TG. The increase in hydrophobicity and rigidity leads to CE maturation which supports the integrity and mechanical resistance of the stratum corneum (SC). Objectives The aim of this work was to develop and validate a novel enzyme activity assay for 12R‐LOX in tape strippings of photo‐exposed (PE) cheek and photo‐protected (PP) post‐auricular SC of healthy Chinese volunteers (n = 12; age 25 ± 3 years). Results A fluorescence‐based assay was developed with ethyl linoleic acid as the substrate and a polyclonal antibody against 12R‐LOX as an inhibitor. The specificity was shown by the lack of effect by a LOX inhibitor (ML351) and an epidermal‐type lipoxygenase 3 (eLOX3) antibody on the acquired 12R‐LOX activity. Reduced 12R‐LOX activity was observed in the outer compared to the inner SC layers. Moreover, dramatically lower activity was shown in the PE vs. PP samples. Furthermore, the enzyme activity has a positive correlation (r = 0.94 ± 0.03) with CE maturity, in particular hydrophobicity, and a negative correlation (r = −0.96 ± 0.01) with transepidermal water loss (TEWL). Conclusion This novel enzyme assay revealed a lower 12R‐LOX activity in tape strippings from PE cheek for the first time. This finding is in line with less mature CEs and higher TEWL compared to PP post‐auricular samples. This study indicates a strong link between 12R‐LOX activity and CE maturation and SC integrity.

BackgroundTerminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R‐lipoxygenase (12R‐LOX) and other enzymes before transglutaminase (TG) attaches ω‐hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross‐linked by TG that has been activated by cathepsin D (CathD).ObjectivesThe primary aim of this work was to demonstrate the impact of relative humidity (RH) during ex vivo CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R‐LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on ex vivo CE maturation was tested at low, optimal and high RH.MethodsThe first and ninth tape strip of photo‐exposed (PE) cheek and photo‐protected (PP) post‐auricular sites of healthy volunteers were selected. Ex vivo CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors.ResultsIrrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for ex vivo CE maturation. The inhibition of 12R‐LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during ex vivo maturation in the presence of TG or 12R‐LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN‐27219. However, a protease inhibitor mix reversed the negative effect of overhydration.ConclusionThe study adds to the understanding of the roles of 12R‐LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC.

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.