bluuebunny/crossref_metadata_2025_split huggingface dataset:
O03 HPV8 leads to Lrig1+ keratinocyte stem cell expansion into the overlying epidermis
Authors: Morgan Huw J , Shorning Boris , Olivero Carlotta , Borgogna Cinzia , Akgul Baki , Gariglio Marisa , Patel Girish K 
Published: July 2023

Abstract
Actinic keratoses (AKs) represent the precursor skin lesions in the pathogenesis of up to 87% of cutaneous squamous cell carcinomas (cSCCs). However, current treatment of AK lesions, at best, lead to only a 25% reduction in cSCC and relies on therapies that target abnormal keratinocytes using topical chemotherapy, immune stimulants or surgical resection. We have previously shown that 44% of AK tissue sample histologies contain koilocytes, wherein genotyping universally identified human papillomavirus 8 (HPV8). To determine the role of HPV8 in the pathogenesis of AKs, we used the K14–HPV8 complete early region (HPV8tg) to generate triple cross-lineage tracing transgenic mice: HPV8:K5-CreERT2:R26R-LoxP-flanked-Brainbow2.1 (Confetti reporter); HPV8:Lrig1-CreERT2:R26R-LoxP-flanked-Brainbow2.1; and HPV8:K15-PGR:R26R-LoxP-flanked-Brainbow2.1. Upon Cre activation, progeny clones exhibit one of four fluorescent colours. Compared to wild-type mice, all HPV8tg mice exhibited an expansion of keratinocytes in the hair follicle infundibulum (77.09 mm3 and 253.43 ± 54.15 mm3) and overlying epidermis. Clones from the Lrig1, but not K15, keratinocyte stem cell population were responsible for keratinocyte expansion of both the hair follicle infundibulum and overlying epidermis. Compared with wild-type mice, HPV8tg mice exhibited greater colony-forming efficiency (1.75 ± 0.69% vs. 6.27 ± 2.41%; n = 3), and, consistent with keratinocyte stem cell expansion, ΔNp63 expression was increased 4.14 ± 2.10-fold (P &lt; 0.05, n = 5). Flow-sorted epidermal clones from wild-type and HPV8tg mice were examined by RNA sequencing (n = 5). Warthin–Starry staining showed that the expanded population had no melanin ‘cap’ in our mouse models and human AK samples. In summary, expression of HPV8 early-region genes is associated with AK, wherein there is an expansion of the Lrig1, but not K15, keratinocyte stem cell population that results in epidermal thickening reminiscent of human AK pathology. These findings suggest that HPV8 results in translocation of hair follicle keratinocyte stem cells into the interfollicular epidermis without melanin production, making these cells susceptible to ultraviolet light transformation.
O06 Exploring host–microbiome–immune interactions using singular and multispecies inoculations in optimized 3D skin models
Authors: McPolin-Hall Esmé , Stephen Abish , Pardieu Claire  , Allaker Robert , Philpott Michae  , Bhogal Ranjit , Pople Jenny , Hannen Rosalind  
Published: July 2023

Abstract
The human microbiome has a fundamental role in skin homeostasis, barrier function and host immune networks. Specifically, a complex and multispecies microbiome population is required for healthy skin. Acute and chronic skin conditions are often associated with imbalances in host–microbiome–immune interactions. Despite recent advances in skin microbiome research, there are limited models for studying these interactions in healthy and diseased skin; therefore, we have developed a model to investigate how different microbiome profiles alter epidermal homeostasis and T-cell responses. Full-thickness three-dimensional (3D) skin models, developed using primary dermal fibroblasts and keratinocytes isolated from adult skin, were colonized with five human microbiome species: Staphylococcus epidermidis (SE), Staphylococcus capitis (SC), Cutibacterium acnes (CA), Malassezia restricta (MR) and Malassezia globosa (MG), in singular or combined inoculations (5M). Significant changes in epidermal thickness were observed in models colonized with single-species inoculations compared with sterile control models; however, no significant changes in epidermal thickness were observed in models colonised with 5M inoculations. The effect of colonization on epidermal differentiation and barrier formation was observed via the expression of K14, K10, E-cadherin and Ki67. Expression of E-cadherin significantly decreased in models colonized with SC, MR and MG compared with sterile control models. Despite significant changes in epidermal thickness, only MG showed a significant decrease in Ki67 expression compared with sterile control models. No significant changes were observed in K10 and K14 expression following microbial inoculation. Overall, 5M inoculation showed no significant changes in biomarker expression or epidermal thickness compared with sterile control models suggesting a combination of microbes supports epidermal homeostasis as observed in vivo. Commensal microbes have been shown to regulate epidermal barrier function via the aryl hydrocarbon receptor (AhR). Increased nuclear expression of AhR was observed in response to SC, CA and 5M, suggesting these microbes have a greater role in AhR activation. To investigate microbiome–immune interactions in the skin, the expression of CD69, cutaneous lymphocyte antigen (CLA) and HLA-DR on T cells was assessed in response to incubating peripheral blood mononuclear cells with conditioned media from inoculated models. SE caused a significant increase in CD3+/CLA+ cells and 5M significantly reduced CD3+/HLA-DR+ cells. RNA sequencing aims to support current analysis and elucidate any more targets to be explored. Our findings suggest different microbial profiles alter epidermal barrier formation and immune responses.
O22 Programmed cell death-1 receptor is a checkpoint in Tbet+ Th17 cells within the tumour microenvironment
Authors: Smith Katie , Lim Jing Xuan , Laurence Arian , Amarnath Shoba 
Published: July 2023

Abstract
Programmed cell death-1 (PD-1) receptor is critical for immune regulation and is expressed on a variety of immune cells, including effector T cells. In tumours, PD-1 engagement dampens immune activation and increases tumour progression. However, it is unclear how PD-1 affects T helper (Th) cell differentiation, namely Th17 cells. Previously, we have demonstrated that PD-1 maintains T regulatory (Treg) cell plasticity, and Tbet+ Th1 cell plasticity to Treg cells in inflammation and cancer. Therefore, it is clear that PD-1 is indispensable in T-cell lineage maintenance, with limited understanding on its role in Th17 differentiation, function and lineage stability. We hypothesized that as PD-1 blockade augments Th17 responses in cancer, PD-1 could control Th17 function and lineage stability within the tumour microenvironment. We established a B16 F10 melanoma model in wild-type (WT) and Pdcd1–/– (PD-1) mice, and isolated Thy1+ tumour-infiltrating lymphocytes (TILs) from tumours. Single-cell analysis was conducted, and protein and gene expression analysed within the RORc cluster. Our results demonstrated that WT and PD1–/– mice possessed RORc-expressing cells within the TILs, with PD1–/– mice showing an increased number of Th17+ TILs, revealing a role for PD-1 in regulating Th17 responses within TILs. We next investigated the RORc cluster for function and found expression of Il17a and Ifng. Of note, we found that Th17 differentiation genes [Dusp2 (P = 0.004); Ddx58 (P = 0.009)] were significantly regulated by PD-1. Although an increase in interleukin (IL)-12RB1 (P = 0.001) and IL-17RB protein (P = 0.004) was noted, suggesting a plasticity towards either Th1 or Th2 phenotypes, a significant downregulation of Tbet (Tbx21, P = 0.003) was observed in the PD1–/– population. These data suggest that PD-1 is required for RORc+ cells to acquire functional plasticity within tumours, thereby highlighting a role for PD-1 in Th17 plasticity. In summary, our study is the first to show the importance of PD-1 in driving Tbet+ Th17 cell differentiation within the TME.
P08 Dissection of the molecular and cellular heterogeneity of dermal fibroblasts in skin fibrosis
Authors: Ahmed Abubkr , Kirk Thomas , Forster Laura  , O’Toole Edel  , Gupta Himadri , Rognoni Emanuel  
Published: July 2023

Abstract
Skin fibrosis, a common scarring condition that currently has no curative treatments, is caused by persistent activation of fibroblasts after tissue injury or inflammation that results in excessive connective tissue deposition, leading to severe disability, organ malfunction and increased morbidity. This study aims to uncover the early and advanced changes in different fibroblast subsets and molecular pathways that are integral to pathological fibroblast activation using a bleomycin-induced skin fibrosis mouse model that recapitulates well the human disease. We have shown that 2 weeks (early) and 4 weeks (advanced) of subcutaneous bleomycin injections result in an increasing severity of skin fibrosis, characterized by increased dermal thickness, collagen deposition, inflammation, and loss of dermal fibroblasts and adipocytes. We performed single-cell RNA sequencing and successfully isolated 14 944 control, 16 344 early and 6860 advanced fibrotic cells. We identified the changes in fibroblast subclusters and gene signatures that are altered across the time course. Using CellChat, we are dissecting the specific signalling crosstalk between different fibroblast subpopulations and other skin cells, promoting a profibrotic phenotype and locking fibroblasts in an activated state. Furthermore, we are intersecting our novel data set with publicly available wound healing transcriptomic data, allowing us to examine how fibroblast subpopulations, behaviour and key signalling pathways vary in healthy wounding healing and pathological scarring. To understand the impact that changes in extracellular matrix biomechanics elicit on profibrotic fibroblast fate, we are correlating our histological and transcriptomic analysis with nanoindentation atomic force microscopy imaging throughout the fibrotic time course. This novel multidisciplinary approach allows us to compare the cellular and molecular changes in early and advanced fibrotic lesions, identifying 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 other organs.
P09 Short-term rho-associated kinase inhibitor treatment promotes primary keratinocyte growth without affecting keratinocyte stem cell properties
Authors: Jayarajan Vignesh , Hall George T , Xenakis Theodoros  , Bulstrode Neil , Moulding Dale  , Castellano Sergi , Di Wei-Li 
Published: July 2023

Abstract
Keratinocyte stem cells (KSCs) can be cultured as ex vivo epidermal sheets and are attractive for cell and gene therapy. However, the growth and expansion of primary keratinocytes (including KSCs) in in vitro culture can be inefficient as KSCs undergo apoptotic cell death, known as anoikis when isolated from skin biopsies and dissociated from their extracellular environment. This further affects the survival and stemness of KSCs in in vitro culture and hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle. However, it risks changing the characteristics of stem cells. In this study, we studied the effect of the Y-27632 ROCKi on cultured primary human keratinocytes. Following a short-term (6 days) ROCKi treatment, we assessed keratinocyte proliferation and differentiation potential and KSC stemness at the molecular-, cellular- and single-cell levels. A significant increase in colony-forming efficiency associated with increased expression of proliferation markers and mitochondrial mass was observed in treated cells vs. untreated cells. Similarly, treated cells showed a distinct transcriptional profile at the single-cell level, but these changes became indistinguishable after discontinuation of ROCKi treatment. Single-cell transcriptome analysis further confirmed that short-term ROCKi treatment did not affect stem cell characteristics. Finally, we showed that ROCKi rapidly activated Akt and upstream of ERK signalling pathways in primary human keratinocytes. In conclusion, rapid proliferation due to short-term ROCKi treatment was transient and reversible, and did not affect the KSC population or their characteristics. We thus present a safer approach for the use of ROCKi in ex vivo clinical applications.
P19 Inducible 14-3-3 sigma/stratifin ablation accelerates malignant progression in HK1.ras/fos-Δ5PTENflx transgenic mouse skin carcinogenesis
Authors: Rawlinson Imogen , McMenemy Carol , Greenhalgh David 
Published: July 2023

Abstract
In human carcinogenesis, given the plethora of pathways associated with 14-3-3σ and their complex interactions, the causality of 14-3-3σ deregulation remains elusive, with both tumour suppressive and oncogenic roles. To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of endogenous 14-3-3σ (stratifin) was analysed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of phosphatase and tensin homolog (PTEN)-mediated Akt regulation (K14.creP/Δ5PTENflx) and via conditional RU486-inducible 14-3-3σ knockout (K14.creP-Δ14-3-3). Consistent with 14-3-3σ roles in the commitment of keratinocytes to differentiate, bigenic HK1.fos/Δ5PTENflx hyperplasia expressed 14-3-3σ in basal layers and paralleled keratin K1 expression which appeared alongside elevated p53/p21 to increase keratinocyte differentiation leading to a keratoacanthoma (KA) aetiology. Trigenic HK1.ras/fos-Δ5PTENflx hyperplasia/papillomas also displayed increased basal-layer 14-3-3σ, suggesting attempts to protect basal layer p53 tumour suppressor gene function, given that 14-3-3σ acts as a chaperone protein to remove/relocate MDM2 for degradation, thus maintaining p53 levels. With time, HK1.ras/fos-Δ5PTENflx papillomas exhibited reduced p53 and increased p-MDM2166 activity in basal layers, which coincided with malignant conversion. Surprisingly, despite this p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCC) and, alongside elevated p21, appeared to limit further malignant progression via inhibiting p-Akt1473 expression. To further assess causality, 14-3-3σ functions were ablated in this model via an RU486-inducible cre/loxP system expressed from a keratin 14 (K14) promoter (K14.creP-Δ14-3-3σ). In bigenic HK1.fos-Δ14-3-3σ, preliminary data suggest loss of 14-3-3σ functions resulted in increased hyperplasia and keratosis similar to that observed in bigenic HK1.fos/Δ5PTENflx hyperplasia. Functional 14-3-3σ ablation in HK1.ras.fos/Δ5PTENflx/Δ14-3-3σflx genotypes apparently failed to accelerate papillomatogenesis, suggesting elements of redundancy in terms of tumour promotion; however, loss of 14-3-3σ facilitated increased p-MDM2166 and p53 loss, resulting in malignant conversion with progression to aggressive SCC. Collectively, these data suggest that 14-3-3σ/stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms, while persistent expression in wdSCCs suggests p53-independent scenarios that may involve p21-mediated Akt1 inhibition. However, if ablated, this limit of early-stage malignant progression is lost and 14-3-3σ loss leads to rapid progression to aggressive, p21-negative SCCs exhibiting uniform p-Akt1473activation.
Can portable glucose and lactate meters be a useful tool in quantifying stress of juvenile Chinook salmon?
Authors: Vaage Benjamin M , Liss Stephanie A , Fischer Eric S , Khan Fenton , Hughes James S 
Published: July 2023

Abstract
Blood plasma analyses can provide researchers, aquaculture facilities and fisheries managers with valuable insights into the physiological state and welfare of fish. For example, glucose and lactate are part of the secondary stress response system, and elevated concentrations are indicators of stress. However, analysing blood plasma in the field can be logistically difficult and typically involves sample storage and transport to quantify concentrations in a laboratory setting. Portable glucose and lactate meters offer an alternative to laboratory assays and have shown to be relatively accurate in fish, but these tools have only been validated for a few fish species. The objective of this study was to investigate if portable meters could be reliably used in Chinook salmon (Oncorhynchus tshawytscha). As part of a larger stress response study, juvenile Chinook salmon (157 ± 17 mm fork length [mean ± standard deviation; SD]) were exposed to stress-inducing treatments and sampled for blood. Laboratory reference glucose concentrations (milligrams per deciliter; mg/dl; n = 70) were positively correlated with the Accu-Check Aviva meter (Roche Diagnostics, Indianapolis, IN) measurements (R2 = 0.79), although glucose values were 1.21 ± 0.21 (mean ± SD) times higher in the laboratory than with the portable meter. Lactate concentrations (milliMolar; mM; n = 52) of the laboratory reference were also positively correlated (R2 = 0.76) with the Lactate Plus meter (Nova Biomedical, Waltham, MA) and were 2.55 ± 0.50 times higher than portable meter. Our results indicate both meters could be used to measure relative glucose and lactate concentrations in Chinook salmon and provide fisheries professionals with a valuable tool, particularly in remote field settings.
Nitrogen fixation in the stag beetle, Ceruchus piceus (Coleoptera: Lucanidae): could insects contribute more to ecosystem nitrogen budgets than previously thought?
Authors: Mifsud Isobel E J , Akana Palani R , Bytnerowicz Thomas A , Davis Steven R , Menge Duncan N L 
Published: July 2023

Abstract
Nitrogen (N) is a key nutrient required by all living organisms for growth and development, but is a limiting resource for many organisms. Organisms that feed on material with low N content, such as wood, might be particularly prone to N limitation. In this study, we investigated the degree to which the xylophagous larvae of the stag beetle Ceruchus piceus (Weber) use associations with N-fixing bacteria to acquire N. We paired acetylene reduction assays by cavity ring-down absorption spectroscopy (ARACAS) with 15N2 incubations to characterize rates of N fixation within C. piceus. Not only did we detect significant N fixation activity within C. piceus larvae, but we calculated a rate that was substantially higher than most previous reports for N fixation in insects. While taking these measurements, we discovered that N fixation within C. piceus can decline rapidly in a lab setting. Consequently, our results demonstrate that previous studies, which commonly keep insects in the lab for long periods of time prior to and during measurement, may have systematically under-reported rates of N fixation in insects. This suggests that within-insect N fixation may contribute more to insect nutrition and ecosystem-scale N budgets than previously thought.
Substantial changes in inflammatory and cardiovascular biomarkers in patients with autonomous cortisol secretion
Authors: Ueland Grethe Å , Methlie Paal , Heie Anette , Meling Stokland Ann-Elin , Dahle Anne Lise , Sævik Åse B , Løvås Kristian , Husebye Eystein S 
Published: July 2023

AbstractObjectiveTo map inflammatory biomarkers in patients with autonomous cortisol secretion (ACS) and overt Cushing syndrome (CS).MethodObservational study including serum from prospectively included patients with ACS (n = 63), adrenal CS (n = 2), pituitary CS (n = 8), and healthy subjects (n = 120). Serum samples were analysed for 92 inflammatory biomarkers using proximity extension assay (OLINK).ResultsCombined, the ACS and CS patients displayed significant differences in levels of 49/92 inflammatory biomarkers (46 increased/3 decreased) compared with healthy controls. No differences in biomarker levels were found between ACS and overt CS, and none of the biomarkers correlated with the degree of hypercortisolism. Postoperative samples were available for 17 patients, median 24 months (range 6–40) after surgery and biochemical curation. There was no significant normalization of the biomarkers postoperatively.ConclusionThere was a systemic rise in inflammatory biomarkers in patients with ACS and CS, not correlated to the degree of hypercortisolism. These biomarkers were not normalized following biochemical cure.
Inhibitory Neurons in Nucleus Tractus Solitarius Are Involved in Decrease of Heart Rate Variability and Development of Depression-Like Behaviors in Temporal Lobe Epilepsy
Authors: Aimaier Guliqiemu , Qian Kun , Cao Huateng , Peng Weifeng , Zhang Zhe , Ma Jianhua , Ding Jing , Wang Xin 
Published: July 2023

AbstractBackgroundDiminished heart rate variability (HRV) has been observed in epilepsy, especially in epilepsy with depressive disorders. However, the underlying mechanism remains elusive.MethodsWe studied HRV, spontaneous recurrent seizures, and depression-like behaviors in different phases of pilocarpine-induced temporal lobe epilepsy (TLE) in mice. Single-cell RNA sequencing analysis was used to identify various nerve cell subsets in TLE mice with and without depression. Differentially expressed gene (DEG) analysis was performed in epilepsy, depression, and HRV central control–related brain areas.ResultsWe found decreased HRV parameters in TLE mice, and alterations were positively correlated with the severity of depression-like behaviors. The severity of depression-like behaviors was correlated with the frequency of spontaneous recurrent seizure. Characteristic expression of mitochondria-related genes was significantly elevated in mice with depression in glial cells, and the enrichment analysis of those DEGs showed an enriched GABAergic synapse pathway in the HRV central control–related brain area. Furthermore, inhibitory neurons in the nucleus tractus solitarius, which is an HRV central control–related brain area, were specifically expressed in TLE mice combined with depression compared with those in mice without depression. A significantly enriched long-term depression pathway in DEGs from inhibitory neurons was found.ConclusionsOur study reported correlations between HRV and epilepsy–depression comorbidity in different phases of TLE. More importantly, we found that HRV central control-related inhibitory neurons are involved in the development of depression in TLE, providing new insights into epilepsy comorbid with depression.