bluuebunny/crossref_metadata_2025_split huggingface dataset:
O02 Neoantigens from actinic keratosis are predicted to be more immunogenic than those from cutaneous squamous cell carcinoma – a strategy for immune escape?
Authors: Thomson Jason , Harwoood Catherine , Strid Jessica  , Healy Eugene , Wang Jun 
Published: July 2023

Abstract
Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). More than 70% of cSCC arise from AKs, but fewer than 0.6% of AKs progress to cSCC. We have shown that the molecular landscapes of AK and cSCC are similar in terms of tumour mutational burden (TMB), copy number burden, mutational signatures and driver gene mutations but differ in levels of intrasample heterogeneity. We have also demonstrated that alterations in signalling pathways, including transforming growth factor-β, may be important for AK-to-SCC progression. However, the pathomechanisms underlying progression are not well characterized and it remains unclear why the majority of AKs do not progress, and most will regress. There is some evidence that differences in the neoantigen landscapes of AK and cSCC may play a role, and a previous study examining neoantigens in AK and cSCC from nine patients was the first to suggest that there is downregulation of the expression of highly immunogenic neoantigens in the immune escape of cSCC. In this study we further explore, in a larger data set, the hypothesis that the neoantigen recognition potential of AK vs. cSCC is a key determinant of AK-to-cSCC progression. We generated exome and transcriptome data from AK (n = 5) and cSCC (n = 15) with matched normal exome data (from blood). Human leucocyte antigen typing was performed using the OptiType algorithm and neoantigens restricted to major histocompatibility complex (MHC) class I were predicted using the NeoPredPipe workflow. Fitness of neoantigens, defined as the overall effectiveness of a neoantigen at stimulating an immune response, was determined by combining measures of MHC binding affinity and T-cell receptor recognition potential for the predicted neoantigen, and an overall neoantigen recognition potential score was derived for each neoantigen. The difference in median total neoantigen burden for AK and cSCC was not statistically significant [11 224 (range 30–17 330) and 1680 (range 3159–67 357), respectively (P = 0.07)]. Neoantigen burden was strongly correlated with TMB in both AK and cSCC. We found that, compared with cSCC, AK had predicted neoantigens with significantly higher mean MHC-binding affinity (P = 0.049), mean T-cell receptor recognition potential (P = 0.0093) and overall neoantigen recognition potential (P = 0.02). Taken together, our results confirm that neoantigens in AKs are predicted to be more effective at eliciting an immune response than those in cSCC. This suggests there may be a selection pressure in AKs for mutations that result in fewer immunogenic neoantigens resulting in immune escape and progression to cSCC. Finally, our results underscore the critical requirement for an intact immune system in prevention of AK-to-cSCC progression.
O11 The human discs large protein (Dlg1) controls connexin 43 (Cx43) plasma membrane localization and gap junctional communication in keratinocytes
Authors: Scott Harry , Dong Li , Srinivasan Sharmila , Stevenson Andrew , MacDonald Alasdair , Massimi Paola  , Banks Lawrence , Martin Patricia , Johnstone Scott , Graham Sheila 
Published: July 2023

Abstract
Gap junctional communication is required for cellular coordination in health and disease. Gap junction channels are composed of connexins and allow direct intercellular ­communication. Connexins also play an important role in processes such as wound healing, with connexin 43 (Cx43) being downregulated at the plasma membrane of cells at the wound edge during the initial stages of the normal wound healing process. Connexins must be located on the plasma membrane in order to form gap junctions; therefore, it is important to identify proteins involved in the regulation of this. Previously, in a study of human papillomavirus (HPV)-positive tumour cells, we reported that the gap junction protein Cx43 is a physiologically relevant binding partner of the human homologue of Drosophila Discs large (Dlg1). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family that is known to control cell shape and polarity, and intracellular trafficking. Here we show, for the first time, that Cx43 interacts with Dlg1 in uninfected keratinocytes in vitro and in normal human epithelial tissues. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with an overall reduction in Cx43 protein levels. The loss of Dlg1 in keratinocytes was further associated with a loss of Cx43 at the plasma membrane and a reduction in intercellular communication. This decrease in Cx43 levels was partially reversed through use of a lysosomal inhibitor; however, a proteasomal inhibitor resulted in no difference to Cx43 levels, suggesting that, in the absence of Dlg1, Cx43 is degraded through the lysosomal but not the proteasomal pathway. This correlated with an increased association between Cx43 and LAMP2, a marker of lysosomes in Dlg1 knockdown cells. In the absence of Dlg1, Cx43 localized to the Golgi but not the endoplasmic reticulum. Although this result could indicate that Dlg1 is required for stable Cx43 membrane location, a study of Cx43 and Dlg1 during the wound healing process in HaCaT cells (a spontaneously immortalized HPV-negative epithelial cell line), showed the proteins co-localizing as they relocated from the plasma membrane to the cytoplasm between 4 and 16 h postwounding before returning to the plasma membrane at 24 h postwounding. This may suggest that Dlg1 is involved in Cx43 trafficking. The levels of both proteins were reduced between 0 and 4 h postwounding and then increased between 4 and 16 h, before returning to normal levels at 24 h postwounding. Taken together, our data suggest that Dlg1 is crucial for promoting plasma membrane localization of Cx43 in keratinocytes.
O15 Using CRISPR-Cas9 to annotate the TYK2 pathway in psoriasis
Authors: Elyoussfi Sarah , Rane Shraddha S , Eyre Stephen  , Warren Richard B 
Published: July 2023

Abstract
Genome-wide association studies (GWAS) have discovered genetic signals associated with psoriasis. This study is the first to validate functionally the psoriasis-associated single nucleotide polymorphism (SNP) rs892085 on TYK2 expression using clustered regularly interspaced short-palindromic repeat Cas9 endonuclease (CRISPR-Cas9) technologies in Jurkat T-lymphoblast cells. Our in-house capture Hi-C data revealed long-range chromatin interactions between specific SNP sets and TYK2. An independent noncoding genetic association with psoriasis was identified about 400 kb upstream of TYK2: rs892085 at 19p13.2. This is a psoriasis-associated risk SNP (P = 3.0 × 10–17). To characterize fully the physical connection, CRISPR-Cas9 genome editing was conducted in T-cell-like Jurkat T-lymphoblast cell lines. We used the CRISPR activator (CRISPR/dCas9-VP64) and CRISPR interference (CRISPR/dCas9-KRAB) systems to alter the transcriptional activity of regions to which they are targeted. We performed reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to identify differential gene expression between modified and control cells. RT-qPCR revealed consistent alteration of TYK2 expression upon inhibition and activation of rs892085. This supports previous observations, where this regulatory region harbouring GWAS-implicated variation operates through direct long-distance contact. To associate genes with disease, the closest or most biological relevant gene is often assigned to a GWAS variant. We have shown that this is not always reliable, with DNA regions looping over large distances to control genes. This provides the first functional validation of the psoriasis-associated SNP rs892085. GWAS have linked TYK2 to psoriasis susceptibility and TYK2 is a recognized drug target with one therapy, deucravacitinib, already approved by the US Food and Drugs Administration. This work demonstrates how risk alleles may work to dysregulate TYK2 signalling in disease. Ultimately, genetic risk scores may be generated to predict how a patient may respond to tyrosine kinase 2 (TYK2) therapy. Functional annotation of loci that perturb the TYK2 pathway will be the key to optimizing TYK2 therapy for the right person at the right time.
O16 The novel roles of laminin α3 in epidermal barrier function and metabolism
Authors: Parzymies Piotr , Jones Eleri Mai , Mayes Andrew  , Messenger David , O’Toole Edel , Caley Matthew  
Published: July 2023

Abstract
The skin serves an important barrier function performed by epidermal lipids, cornified epithelial cells and cell–cell adhesions. This function is compromised in old age and exhibits certain abnormalities such as flattened dermal–epidermal junction or improper lipid metabolism, and is also associated with mitochondrial dysfunction. The levels of a basement membrane protein laminin 332 have been shown to decrease as the skin ages. We have previously demonstrated that loss of laminin 332 leads to an upregulation of genes involved in cholesterol biosynthesis and is associated with an altered skin lipid profile. We showed that this change is caused by an abnormal intracellular cholesterol transport that is actin-dependent. To further elucidate the mechanism around cholesterol transport, we have identified five proteins of interest for further investigation. We have shown that two of those proteins, NPC2 and Myo5b, are decreased with the loss of the α3 laminin 332 subunit in laminin α3 knockdown nTERT keratinocytes vs. shC controls. Interestingly, the NPC2 binding partner, NPC1, appears to be increased in the laminin α3 knockdown nTERT keratinocytes. Our analysis has also demonstrated that e-cadherin, a crucial cell–cell adhesion protein, as well as several differentiation markers, are altered with the loss of laminin α3. We have carried out laminin 332 recovery experiments in two (2D) and three dimensions, which showed that the addition of recombinant laminin 332 restores the proper levels of myo5b and e-cadherin, suggesting a potential therapeutic effect. We have also performed proteomic, lipidomic, and transcriptomic studies in 2D, which demonstrated statistically significant changes (Q-value ≤ 0.01) in proteins involved in actin and microtubule regulation, cell–cell adhesion and showed unexpected mitochondrial metabolic changes associated with the loss of laminin α3. These findings show that laminin α3 plays numerous, previously unreported roles in epidermal lipid transport, cell–cell adhesion, differentiation and metabolism, and suggests a potential therapeutic role of the protein in skin ageing.
O19 CYLD cutaneous syndrome tumours demonstrate increased NF-κB signalling and diminished collagen organisation.
Authors: Inns Joseph , Rajan Neil 
Published: July 2023

Abstract
CYLD is a tumour-suppressor gene encoding a deubiquitinase, targeting K63- and M1-linked ubiquitin chains and thereby influencing a variety of cellular processes. Loss of function in patients with CYLD cutaneous syndrome (CCS) results in the formation of benign cutaneous cylindromas, spiradenomas and trichoepitheliomas. These CCS skin tumours originate from the hair follicle and appear from adolescence onwards. We used an unbiased proteomics approach to analyse CCS tumour and control samples (n = 8). Label-free quantification of 627 proteins revealed significant changes in 139 proteins with a fold change &gt; 1.75 and P-value &lt;0.05, between control and tumour samples. Gene Ontology analysis identified upregulation of nuclear factor-κB (NF-κB) and immune signalling proteins alongside an increased prevalence of spliceosome proteins in tumour samples. Collagen 7, collagen 4, agrin and thrombospondin-1 were increased in tumour samples, providing, for the first time, an unbiased set of extracellular matrix (ECM) proteins generated by CCS tumour cells. Conversely, CCS tumour samples demonstrated a decreased abundance of proteins associated with collagen organization and fibril formation compared to normal skin. Further analysis of CCS tumours through immunoblotting revealed a reduction of collagen I, while collagen VII was highly upregulated in both tumour lysate and secretome. Our findings indicate that loss of CYLD function impacts on cellular signalling pathways such as NF-κB signalling, and is associated with hypersecretion of select ECM proteins.
O21 Single-cell RNA sequencing and proteomic analysis of dermal fibroblasts cultured from female scalp identifies significant alterations in cell populations with age
Authors: Baker Richard , Williams Rachael , Westgate Gillian  , Pawlus Alison , Zguris Jeanna , Thornton Julie 
Published: July 2023

Abstract
We have previously reported that significant in situ detrimental changes occur in the female scalp with age. We also reported that primary cultures of female scalp dermal fibroblasts (DF) significantly increase the secretion of proteins such as cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5), secreted protein acidic and cysteine rich (SPARC) and smooth muscle actin (ACTA2). This highlights changes in DF cellular identity with age, altering the immediate hair follicle environment, presumably driving hair ageing. To gain a better understanding of age-related molecular and functional changes in DFs, we compared both the transcriptional and proteomic profile of primary cultures of scalp DFs (passage &lt; 3) from female donors (n = 8) aged 21–77 years. Transcriptional profiles were analysed via 10× Genomics scRNAseq. Data profiles were generated by evaluation of cellular lysates by liquid chromatography mass spectrometry proteomics and compared by MS1 precursor intensities using PEAKS software. A total of 16 166 DF cells from young (n = 3) and older (n = 3) donors were sequenced. Across the two populations, 11 transcriptionally distinct clusters were detected, highlighting the heterogeneity in DFs. The percentage of cells in these clusters changed with age, and gene set enrichment analysis ascertained a significant increase of cells in cluster 2, which represented a higher number of gene sets related to fibrosis, senescence, collagen synthesis and degradation. Proteomic analysis of DF cellular proteins (four donors in each age group) identified 11 significantly differentially expressed proteins between the two age groups. Two proteins (serine/threonine-protein kinase 38 and rho GTPase activating protein 21) were significantly reduced in the older cohort, while nine proteins (smoothelin, retinol dehydrogenase 5 tetranectin, oestradiol 17-beta-dehydrogenase 11, CD14, carboxypeptidase M, gamma-enolase, plexin-B1 and endoplasmic reticulum membrane protein complex subunit 4) were significantly elevated in the older cohort. Gene Ontology and STRING analysis of the data revealed modifications to key pathways with age (e.g. collagen and elastic fibre formation, autophagy, ubiquitination and metabolic markers). This study highlights the loss of scalp DF cell signature and identity with age. We hypothesize that these changes impact on the ability of the scalp dermal environment to support and maintain healthy hair follicles, contributing to the age-related changes observed in female hair growth.
P06 Functional MDM2 ablation in HK1.ras/fos-Δ5PTEN transgenic mice results in an elevated, p53-mediated block of carcinogenesis that depends upon p53 expression
Authors: Moula Sofia , Turnbull Gavin , Greenhalgh David  
Published: July 2023

Abstract
MDM2 is a ubiquitin ligase that targets p53 for degradation and the efficacy of functional MDM2 knockout to protect p53 levels was shown to inhibit benign papilloma formation in HK1.ras transgenic mice. In skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of phosphatase and tensin homolog (PTEN)-mediated Akt regulation, p53 loss was a consistent theme leading to well-differentiated squamous cell carcinoma (SCC). Analysis of endogenous MDM2 status in HK1.ras/fos-Δ5PTENflx hyperplasia and papillomas showed that activated p-MDM2166 activity was initially confined to suprabasal layers with resultant elevated basal layer p53. However, with time, HK1.ras/fos-Δ5PTENflx papillomas exhibited reduced p53 levels in basal keratinocytes associated with increased p-MDM2166 activity, which coincided with malignant conversion, suggesting that suprabasal-to-basal increases in p-MDM2166 downregulated p53 giving a susceptibility to malignant conversion. Therefore, to assess whether removal of this MDM2 ability to degrade p53 could inhibit carcinogenesis in HK1.ras/fos-Δ5PTENflx mice, MDM2 exons 5 and 6, which bind p53 and chaperone for ubiquitination, were removed via a Cre/lox-P system driven by a keratin K14 promoter following treatment with topical RU486 (K14cre-Δ5/6MDM2flx/flx). RU486 treatment of HK1.ras/fos-Δ5PTENflx-Δ5/6MDM2flx/flx mouse skin resulted in a complete block of carcinogenesis in all animals for over 16 weeks due to elevated p53 expression. However, preneoplastic hyperplasia appeared to be relatively unaffected, despite excess p53 expression. In approximately 10–15% of animals, HK1.ras/fos-Δ5PTENflx-Δ5/6MDM2flx/flx mice developed overt tumours by 5–6 months. A similar finding was observed in HK1.fos-Δ5PTENflx keratoacanthoma (KA) aetiology, as here the early p53 expression in HK1.fos-Δ5PTENflx-Δ5/6MDM2flx/flx hyperplasia mediated by MDM2 loss also blocked KA aetiology. To assess whether this was specific to MDM2/p53 interactions, inducible p53 ablation was introduced into the HK1.fos-Δ5PTENflx KA model. This confirmed inhibition was specific to p53 expression, as all HK1.fos-Δ5PTENflx/p53 flx/flx tumours rapidly progressed to malignancy, regardless of MDM2 genetic status. Thus, MDM2 inhibition can limit carcinogenesis via maintaining p53 levels; however, this inhibition can be circumvented via p53 loss and, moreover, MDM2 inhibition would be counterproductive in SSC contexts that expressed gain-of-function p53 mutations.
P26 Characterization of skin barrier defects in atopic dermatitis patients using attenuated total reflection Fourier transform infrared spectroscopy
Authors: Williams Samuel , Danby Simon 
Published: July 2023

Abstract
Atopic dermatitis (AD) is a common disorder characterized by skin barrier defects often measured by traditional biophysical tools that give little molecular insight into disease pathogenesis. The objective was to employ in vivo infrared spectroscopy as a complimentary technique to biophysical measurements to analyse changes in the chemical composition of the stratum corneum (SC) in relation to AD severity. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy measurements were collected on the forearm alongside transepidermal water loss (TEWL), skin surface pH and capacitance in a cohort of 75 participants, stratified by AD severity. Skin tape stripping was performed to allow measurements at greater SC depth. The five common filaggrin (FLG) loss-of-function mutations were genotyped. Skin hydration, TEWL and redness were all affected by AD severity with molecular analysis showing concomitant changes in lipid structure, and the amount of lipid, polyamide and carboxylate. Correlation analysis showed associations between Eczema Area and Severity Index and lipid structure (r = –0.58), as well as amount of carboxylate (r = –0.49). Principal component analysis showed spectra collected on participants with AD lesions or FLG mutations clustered away from healthy and nonlesional skin sites. Our findings showed that ATR-FTIR spectroscopy is able to characterize structural skin barrier defects in people with AD vs. controls. Spectral peak analysis showed associations between defective lipid and natural moisturizing factor (NMF) components with worsening disease severity. Through combined assessment of peaks associated with lipid structure and carboxylate amount, we have shown the potential of ATR-FTIR in identifying filaggrin deficiency, through either genetic predisposition (FLG mutations) or through inflammatory action, which is related to the presence of NMFs in the SC, skin hydration and overall severity of AD.
P31 Dissecting the epigenetic and transcriptional regulators in keloid scars
Authors: Tekkela Stavroula , Drudi Elena M , Shaw Tanya , Philpott Mike , O’Toole Edel , Rognoni Emanuel 
Published: July 2023

Abstract
Keloids are a type of fibroproliferative scar that progressively grow beyond the original wound site. Currently, there are no treatments to halt or revert keloid scarring, emphasizing the great unmet clinical need and incomplete mechanistic understanding. They occur due to genetic, epigenetic and environmental abnormalities. However, the detailed molecular signatures that promote this pathogenic phenotype are still unknown. Fibroblasts are best characterized for their role in depositing and remodelling the extracellular matrix and presumed to be one of the main cells involved in keloid. This study investigates if keloid fibroblasts are epigenetically locked in an activated state, and if manipulation of this state can reverse the persistent keloid phenotype. To investigate the epigenetic and genetic changes in keloid fibroblasts, we performed ATACseq and RNAseq on a patient-matched set of keloid and normal fibroblasts. ATACseq is a high-throughput sequencing method that defines chromatin-accessible regions in which transcription factor binding sites can be mapped that can regulate gene expression. Differences in chromatin accessibility were found to be closely associated with gene expression differences in keloid fibroblasts. ATACseq analysis revealed accessible regions at genes that are implicated in pathways, including transcriptional regulation, embryonic development, calcium binding, collagen remodelling and apoptosis. Identified candidates driving keloid formation will be validated in keloid skin sections and functionally analysed in two- and three-dimensional culture models after pharmacological or genetic manipulation. These findings will support the development of fibroblast-targeted therapies for keloid scars and possibly other connective tissue disorders.
Time-based reperfusion in haemodynamically unstable pulmonary embolism patients: does early reperfusion therapy improve survival?
Authors: Zuin Marco , Piazza Gregory , Barco Stefano , Bikdeli Behnood , Hobohm Lukas , Giannakoulas George , Konstantinides Stavros 
Published: July 2023

Abstract
High-risk pulmonary embolism (PE) is associated with significant morbidity and mortality. Systemic thrombolysis remains the most evidenced-based treatment for haemodynamically unstable PE, but in daily clinical practice, it remains largely underused. In addition, unlike acute myocardial infarction or stroke, a clear time window for reperfusion therapy, including fibrinolysis, for high-risk PE has not been defined either for fibrinolysis or for the more recently incorporated options of catheter-based thrombolysis or thrombectomy. The aim of the present article is to review the current evidence supporting the potential benefit of earlier administration of reperfusion in haemodynamically unstable PE patients and suggest some potential strategies to further explore this issue.