bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis
Authors: Khanabdali Ramin , Mandrekar Michelle , Grygiel Rick , Vo Phuoc-An , Palma Carlos , Nikseresht Sara , Barton Siena , Shojaee Mozhgan , Bhuiyan Sadman , Asari Kartini , Belzer Susan , Ansari Khairul , Coward Jermaine I , Perrin Lewis , Hooper John , Guanzon Dominic , Lai Andrew , Salomon Carlos , Kershner Kevin , Newton Christine , Horejsh Douglas , Rice Gregory 
Published: May 2024

Abstract
Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.
Isolation of short RNAs with homogeneous 3′-ends using quaternary-amine anion exchange chromatography
Authors: Li Zixian , Bilic Mia , Nagar Bhushan 
Published: May 2024

Abstract
Visualizing RNA–protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single-nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein-binding partner.
O22 Unravelling the pathophysiology of KLICK syndrome to identify therapeutically targetable pathways
Authors: Cherry Hannah , Rastrick Joe , Jacków Joanna , Parsons Maddy , McGrath John 
Published: May 2024

Abstract

Introduction and aims
Keratosis linearis with ichthyosis congenita and sclerosing keratoderma (KLICK) syndrome is an ultrarare genodermatosis associated with a homozygous pathogenic variant, c.-95delC in POMP, which encodes for proteasome maturation protein. Clinical manifestations include linear hyperkeratotic papules in the flexures, palmoplantar keratoderma and pseudoainhum. The pathophysiology of KLICK syndrome is poorly understood, resulting in nonspecific, limited treatment options. The aim of this work is to characterize KLICK syndrome at the cellular and molecular level, with the utilization of transcriptomic techniques for potential drug repurposing.

Methods
A lesional skin biopsy was obtained, following informed consent, from a 32-year-old female patient with KLICK syndrome. Histology and immunostaining were used to characterize the disrupted skin architecture. RNA sequencing was used to identify key dysregulated pathways and the L1000FWD reverse transcriptomics tool enabled the generation of a shortlist of potential therapeutics for KLICK syndrome. CRISPR editing techniques and induced pluripotent stem cell (iPSC) reprogramming Methods were used to generate cellular based models carrying the pathogenic POMP variant in order to support evaluation of the shortlisted therapeutic options.

Results
KLICK syndrome skin histology featured hyperkeratosis, hypergranulosis and delayed cellular flattening. Consistent with pathological features, pathway analysis of transcriptomic data revealed keratinocyte differentiation and epidermis development as key upregulated pathways in KLICK syndrome. Interestingly, the Panther pathway analysis tool suggested dysregulation in epidermal growth factor receptor (EGFR) signalling, and reverse transcriptomics identified erlotinib, an EGFR inhibitor, as a potential therapeutic treatment for KLICK syndrome. HaCaT keratinocytes were successfully edited using CRISPR-Cas9 to carry the KLICK patient POMP variant and a KLICK patient iPSC line was generated to support in vitro KLICK syndrome modelling.

Conclusions
KLICK syndrome is a hyperproliferative inflammatory skin disorder associated with a pathogenic variant in POMP. Reverse transcriptomics is a useful tool for shortlisting drugs with biologically relevant mechanisms of action with potential for treatment of rare genetic diseases such as KLICK syndrome.
P01 Remodelling of the dermoepidermal junction occurs in intrinsically aged Far East Asian skin without significant epidermal atrophy
Authors: Lim Zoe , Lunny Declan , Bonnard Carine , Langton Abigail , Watson Rachel 
Published: May 2024

Abstract

Introduction and aims
Ageing is characterized by a gradual decline in physiological function and is influenced by both intrinsic genetic and extrinsic lifestyle and environmental factors. However, much of our knowledge of cutaneous ageing comes from the study of White populations. We have previously shown that the organization of young healthy skin is ethnicity-dependent and that altered epidermal morphology in White volunteers correlates with loss of biomechanical function in the skin. In this study, we used epidermal morphometry to determine whether parameters associated with intrinsic skin ageing in White skin are also observed in Far East Asian skin.

Methods
Archival skin samples were obtained from the Asian Skin Biobank [young (3–21 years), N = 38, 37 male samples (Chinese, n = 16; Malay, n = 16; other Far East, n = 6); aged (61–86 years), N = 7, one male sample (Chinese, n = 6; Malay, n = 1)] and stained with haematoxylin and eosin prior to image analysis for dermoepidermal junction (DEJ) convolution and measurement of epidermal thickness. Weigert’s resorcin-fuchsin stain was used to ensure samples were photoprotected.

Results
Weigert’s resorcin-fuchsin stain revealed that all samples were photoprotected, with no evidence of solar elastosis. We next compared whether geographic ancestry of our local Far East Asian population impacted epidermal morphology in young skin; neither DEJ convolution nor epidermal thickness were impacted by geographic ancestry. However, a significant flattening of the DEJ was observed between young and aged samples (P &lt; 0.01). Interestingly, no significant difference in epidermal thickness was observed as a function of age.

Conclusions
While intrinsically aged Asian skin undergoes the same flattening of the DEJ that is seen in White populations, epidermal thickness does not significantly alter; this is in stark contrast to intrinsically aged White skin, where epidermal atrophy is observed. Hence, the trajectory of intrinsic cutaneous ageing may be ethnicity-dependent, providing further evidence that studies of skin from different ethnicities are necessary.
P02 Accelerated ageing of hair follicles in androgenetic alopecia
Authors: Limbu Summik , Farhat Kim , Redmond Leah , Farjo Nilofer , Farjo Bessam , Kemp Paul , Higgins Claire 
Published: May 2024

Abstract

Introduction and aims
Androgenetic alopecia (AGA) is defined by hair loss from the frontal, parietal and vertex regions of the scalp; however, occipital follicles are spared. Hair follicles in AGA undergo miniaturization, which is the transition of terminal follicles into vellus follicles. Similarly with age, the diameter of the hair shaft decreases with loss of pigmentation, which is also seen in miniaturized follicles. To investigate whether frontal follicles have accelerated ageing compared with occipital follicles, we looked at methylation levels of genes associated with ageing. As the thickness and pigmentation of hair fibre are influenced by the dermal papilla (DP), we conducted microarray analysis of frontal and occipital DP to establish whether accelerated ageing of follicles is driven by DP.

Methods
The biological age of frontal and occipital follicles was calculated using methylation status of ELOVL2, ASPA, EDARADD and PDE4C (n = 3). For microarray analysis, DP was microdissected from the frontal (FDP) and occipital (ODP) follicles from patients undergoing hair transplantation surgeries to treat AGA (n = 4).

Results
Frontal follicles were found to have significantly higher biological age compared with patient-matched occipital follicles suggesting accelerated ageing of follicles in AGA. Transcriptomic analysis revealed significant increase in expression of several genes in FDP and notably EGR1, HSPB1, DNAJB1 and APOD, which have been shown to be upregulated in the DP of aged mice compared with young mice. We found downregulation of TRPS1 in FDP, which represses EGR1. Mutations in TRPS1 underlie trichorhinophalangeal syndrome which is associated with vellus-type hair.

Conclusions
Our transcriptional and molecular analysis revealed that FDP and frontal follicles have an advanced biological age relative to ODP. This increased age is also reflected in the transcriptional signature of FDP. We hypothesize that these changes are in part due to regulation of signalling by the transcriptional repressor TRPS1, whose expression is decreased in FDP.
P07 Mutagenicity of narrowband ultraviolet B
Authors: Fowler Joanna , Coltart George , Sood Roshan , Lai Chester , Nadarajah Noeline , Jones Philip , Healy Eugene 
Published: May 2024

Abstract

Introduction and aims
Exposure to ultraviolet (UV) radiation causes DNA mutations in skin and development of skin cancer. Despite this, UV radiation in the form of narrowband UVB is commonly used by dermatologists to treat skin diseases such as psoriasis. However, the mutagenicity of narrowband UVB in human skin is unknown. In this study, we investigated the mutation burden resulting from a course of narrowband UVB in patients with psoriasis.

Methods
Skin biopsies were taken from uninvolved skin of sixteen patients with psoriasis before and after a treatment course of narrowband UVB. Following microdissection, DNA was extracted from the epidermis and sequenced using nanorate sequencing (NanoSeq), which detects mutations in single molecules of DNA with an error rate of fewer than five errors per billion base pairs. Whole-genome sequencing of blood DNA was employed as germline control. Comparison of mutations in prenarrowband UVB and postnarrowband UVB skin samples allowed quantification of mutation burden and mutational signatures owing to narrowband UVB.

Results
There was a significant increase in the mutation burden in skin following the narrowband UVB treatment course, with UV radiation signatures SBS7a, SBS7b and DBS1 documented in both nonsun-exposed skin and frequently sun-exposed skin samples. Comparison of the magnitude of the mutation burden resulting from narrowband UVB to that in keratinocyte cancers provided insight into the mutagenicity of narrowband UVB in relation to the potential numbers of narrowband UVB courses over a lifetime that might lead to skin cancer development.

Conclusions
Narrowband UVB treatment for psoriasis Results in significant numbers of UV-radiation-induced mutations in human skin.
P14 Characterizing the hair follicle–nervous system connection
Authors: Rhodes Anna , Agramunt Julià , Limbu Summik , Laufer Rimma , Jiménez Francisco , Williams Greg , Knight Leigh , Hashemi Parastoo , Higgins Claire 
Published: May 2024

Abstract

Introduction and aims
The human hair follicle (HF) is a miniorgan in the skin that possesses a rich microenvironment composed of a complex assembly of sensory nerves that surround it. Recent studies have explored the effects of this perineural niche on the HF, but the influence of HF on the nervous system from a folliculocentric and skin-centric perspective remains underexplored. To explore the potential bidirectionality of the HF–nervous system connection in the context of nerve development, we asked whether the HF influences the differentiation and maturation of sensory neuron subtypes around it through the release of distinct neurotransmitters and neurotrophic factors.

Methods
To first identify which cell types might be involved in directing sensory neuron development, we reanalysed available skin and HF single-cell RNA sequencing (scRNA-Seq) datasets. Next, to evaluate neurotransmitter release from these cell types, we used fast-scan cyclic voltammetry (FSCV). Lastly, to interrogate the role of the HF on directing neuronal development in vitro, we used induced pluripotent stem cell-derived sensory neurons as a model for developing nerves.

Results
We found that a higher percentage of KRT14- (epithelial) and PDGFRA-expressing (mesenchymal) cells in the HF coexpressed genes encoding specific neurotrophic factors and enzymatic machinery for neurotransmitter production compared with equivalent cells in the skin, suggesting that the HF possesses a greater capacity to signal to the nervous system compared with interfollicular skin. Using FSCV, we then found that follicular outer root sheath and directionally selective cells are capable of releasing neurotransmitters such as serotonin and histamine.

Conclusions
We postulate that HF-derived neurotransmitters are important for aspects of sensory neuron development such as axonal growth, as serotonin has previously been shown to be an attractive guidance cue for neuronal growth cones. Overall, our work sheds light on the HF–nervous system connection and how the HF may regulate its microenvironment in the skin.
P17 Dissection of the molecular and cellular heterogeneity of dermal fibroblasts in skin fibrosis
Authors: Ahmed Abubkr , Kirk Thomas , O’Toole Edel , Rognoni Emanuel 
Published: May 2024

Abstract

Introduction and aims
Skin fibrosis, a common fibrotic condition that currently has no specific treatments or cures, is caused by persistent activation of skin fibroblasts after tissue injury that Results in excessive scarring, can cause severe disability and seriously affect morbidity. This study aims to uncover the specific fibroblast subsets and molecular pathways that are integral to this sustained fibroblast activation using a bleomycin-induced skin fibrosis mouse model that accurately recapitulates the human disease.

Methods
We have successfully shown that 2 weeks (early) and 4 weeks (advanced) of bleomycin injections Results in an increasing severity of skin fibrosis characteristics. Following this treatment, we performed single-cell RNA sequencing (scRNA-Seq) using control, early and advanced fibrotic tissue and identified fibroblast subclusters and specific gene signatures that are altered across the time course and may play a role in the fibrotic phenotype we observe. The use of advanced scRNA-Seq analysis techniques such as SCENIC and regulon analysis has allowed us to further characterize the changes in fibroblast states during fibrosis.

Results
Our scRNA-Seq data show a loss of fibroblast heterogeneity alongside the emergence of two main subclusters with distinct gene signatures in the advanced fibrotic state. While proliferation is minimal, RNA velocity analysis indicates that the increase in cell count is due to the convergence of neighbouring fibroblast subclusters. The pronounced profibrotic gene expression point to its key role in driving skin fibrosis and we are now exploring the therapeutic potential of chemically or genetically targeting this subcluster.

Conclusions
Our novel skin fibrosis time course scRNA-Seq dataset allows us to compare the cellular and molecular changes in early and advanced fibrotic lesions in order to identify the key fibroblast subpopulations, molecular regulators and connective tissue alterations promoting fibrotic tissue repair. Our findings will support the development of new fibroblast-targeted fibrosis therapies in skin and potentially in other organs.
P29 Long-read sequencing provides unprecedented insight into the skin and wound microbial landscape
Authors: Smythe Paisleigh , Wilkinson Holly 
Published: May 2024

Abstract

Introduction and aims
It is becoming increasingly evident that the microbial inhabitants of our skin play a key role in cutaneous health and disease. However, our understanding of the contribution of the complex skin microbiome to wound healing outcome remains incomplete. This is, in part, due to the historic challenges around traditional sampling and profiling Methods, which often fail to capture the abundance and diversity of the skin microbiome. Recent advances in microbiome sequencing technologies now enable more comprehensive characterization of skin bacterial composition and host-microbe interactions. Thus, the aim of this study was to utilize a novel long-read sequencing platform to provide species-level microbiome characterization and deliver important insight into the drivers of pathological skin repair.

Methods
Microbial DNA samples were collected from the skin and wounds of healthy volunteers and patients with chronic wounds (n = 40 per group). Long-read nanopore sequencing was used to profile microbiome samples, while RNA-Seq enabled generation of donor transcriptomic profiles. A multifaceted approach was used to integrate these datasets to investigate the complex interplay between host skin pathology and the microbiome.

Results
We first identified perturbations in the skin microbiome of patients with chronic wounds, which was significantly less diverse than healthy skin and colonized with key bacteria associated with pathogenicity. These alterations in the skin microbiome also correlated with transcriptomic changes, such as upregulation of genes associated with inflammation, stress and poor healing. Interestingly, long-read sequencing data displayed poor correlation with routine clinical microbiology swabs, while specific clinical characteristics (e.g. glycaemic control and wound duration) were strongly linked to wound microbiome composition.

Conclusions
Collectively, these data provide crucial new insight into the skin and wound microbial landscape, demonstrating a potential role for specific wound bacteria in modulating pathological skin repair.
P30 Exposure to particulate matter induces structural and pigmentary changes to human skin
Authors: Deyvapriya S , Halai Poonam , Kiss Orsolya , Mambwe Bezaleel , Watson Rachel , Langton Abigail 
Published: May 2024

Abstract

Introduction and aims
The majority of the world’s population reside in places with high levels of air pollution. The impact of air pollution on human health has been extensively studied and demonstrates a significant correlation between particulate matter (PM) and risk of cardiovascular and respiratory diseases. However, little is known about the impact of air pollution on human skin health. Here, we develop an ex vivo skin model to investigate cutaneous changes induced by the topical application of PM.

Methods
PM (0, 0.5, 3, 6, 10, 20 and 40 μg mL−1) was topically applied to Genoskin® NativeSkin Access® (Genoskin, Salem, MA, USA) human ex vivo skin samples of White European ancestry (n = 3) for 8 days, with reapplication on day 4. Resultant samples were assessed using haematoxylin and eosin staining (for morphometric analyses), immunofluorescence for Ki67 and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) (to determine cellular turnover), while pigmentary changes were analysed by colourimetry, Warthin–Starry (WS) staining and immunohistochemistry for the melanin markers tyrosinase (TYR), gp100 and c-kit.

Results
Morphological assessment revealed that epidermal thickness was significantly reduced following exposure to higher doses of PM (r = −0.9163, P &lt; 0.01). In addition, there was reduced epidermal cell proliferation (Ki67) and increased apoptosis (TUNEL) at higher doses of PM. Exposure to PM resulted in a dose-dependent decrease in individual typology angle, which is indicative of skin colour becoming darker (r = −0.7895, P &lt; 0.05). Furthermore, WS staining demonstrated significantly increased epidermal melanin (r = 0.9583, P &lt; 0.01) and significantly increased expression of melanin markers TYR (P &lt; 0.01), gp100 (P &lt; 0.001) and c-kit (P &lt; 0.01) in response to PM exposure.

Conclusions
This study confirms that PM induces pigmentary changes to ex vivo human skin by increasing epidermal melanin. Increased expression of c-kit in response to PM exposure suggests that increased pigmentation occurs owing to the upregulation of the microphthalmia-associated transcription factor signalling pathway. Further investigation of the mechanistic drivers of increased pigmentation is warranted.
P32 Melanin confers protection against ultraviolet radiation-mediated mitochondrial DNA damage in skin of colour
Authors: Mambwe Bezaleel , Langton Abigail 
Published: May 2024

Abstract

Introduction and aims
Melanin is a photoprotective pigment that effectively absorbs and scatters solar ultraviolet radiation (UVR) affording protection against nuclear DNA photodamage. In lightly pigmented skin, UVR-mediated damage of cellular DNA is not limited to nuclear DNA; mitochondrial DNA (mtDNA) is also susceptible to UVR-induced damage leading to an increased frequency of deletions and mutations. However, the protective role of melanin against UVR-induced mtDNA damage is unknown. Therefore, we sought to determine whether melanin affords protection against UVR-induced mtDNA damage.

Methods
Using Genome Analysis Toolkit best practices, mtDNA was extracted from photoexposed forearm and photoprotected buttock of Black African (n = 3) and White Northern European (n = 3) volunteers and sequenced to identify somatic mutations.

Results
Comparison of photoexposed forearm with photoprotected buttock identified a total of 21 somatic mutations, including single-nucleotide transitions, deletions and additions. Of these, three somatic mutations were observed in Black skin, whereas 18 were identified in White skin (P &lt; 0.01). Analysis of single-nucleotide transitions revealed a total of 13 variations and of these, two were identified in Black skin and 11 in White skin. Interestingly, of the somatic mutations observed in White skin, there was enrichment of the cytosine to thymine (C&gt;T) transitions in all volunteers. In contrast, photoexposed Black skin had a thymine to guanine transition at position 414 (T414G) identified for two of the three Black volunteers.

Conclusions
Here we identified that Black skin exhibited fewer mtDNA mutations compared with White skin; enrichment of C&gt;T transitions in White skin is consistent with a previously reported UVR-signature of mtDNA damage, whereas the T414G transition identified in Black photoexposed skin has previously been suggested as a mitochondrial biomarker of photoageing. Our study suggests that melanin confers protection against UVR-mediated mtDNA damage and that the mtDNA damage signature differs between skin types.