bluuebunny/crossref_metadata_2025 huggingface dataset:
High Power Targetry R&D and support for future generation accelerator
Authors: Pellemoine F. , Ammigan K. , Barbier C. , Yonehara K.
Published: July 2023

A high-power target system is a key beam element to complete future High Energy Physics (HEP) experiments. The target endures high power pulsed beam, leading to high cycle thermal stresses/pressures and thermal shocks. The increased beam power will also create significant challenges such as corrosion and radiation damage that can cause harmful effects on the material and degrade their mechanical and thermal properties during irradiation. This can eventually lead to the failure of the material and drastically reduce the lifetime of targets and beam intercepting devices.

Designing a reliable target is already a challenge for MW class facilities today and has led several major accelerator facilities to operate at lower power due to target concerns. With present plans to increase beam power for next generation accelerator facilities in the next decade and the multi-year time-scale to acquire the knowledge on material behavior under such extreme environment, timely R&D of robust high-power targets is critical to fully secure the physics benefits of ambitious accelerator power upgrades.

The next generation of high-power targets for future accelerators will use more complex geometries, novel materials, and new concepts allowing better high heat flux cooling methods. Advanced numerical simulations need to be developed to satisfy the physical design requirements of reliable beam-intercepting devices. In parallel, radiation hardened beam instrumentation irradiation methods for high-power targets must be further developed. Additional irradiation facilities are needed since only a few facilities worldwide offer beams for target testing, and the beam provided may not be appropriate for the specific facility or project.

Thus, a comprehensive research and development program must be implemented to address the challenges that multi-MW targets face.
Interface study on the effect of oxygen/nitrogen ratio in Ni/Ti multilayer deposited by reactive sputtering
Authors: Zhao Shinuan , Zhu Jingtao , Yang Zehua , Zhu Yunping , Sun Hang , Zhao Li
Published: July 2023

Since the growth morphology along and perpendicular to the interface is important for supermirror applications, the dependence of this on the reactive gas has been investigated in Ni/Ti multilayers prepared by reactive magnetron sputtering with variable O2/N2 ratios. The interface properties are characterized by GIXRR, XDS, and TEM measurements. Compared to the case without O2, the presence of 20% O2 in the deposition of Ni layers contributes to smooth and abrupt interfaces. It also suppresses the accumulation of interfacial roughness with the increasing number of layers. However, the abundant oxygen content results in a striking degradation of interface quality associated with the crystallization evolution. Moreover, the lateral correlation length of interfacial roughness exhibits a consistent tendency with the grain size as the oxygen content increases. Following the XPS depth profiles, although N2 and O2 gases were applied in the Ni layer deposition, the N and O were only detected in the Ti layers as the compound for the high chemical activity of Ti. The elemental form in the Ni layers corresponds to the crystalline structure inferred by XRD measurements.
Sensitivity and specificity of thromboelastography for hyperfibrinolysis: Comparison of TEG 5000 and TEG 6S CK LY30 systems
Authors: Fuja Christine , Carll Timothy C , Mikrut Krzysztof , Wool Geoffrey D
Published: July 2023

Objectives

The sensitivity and specificity of clot lysis at 30 minutes after maximum clot strength (LY30), as measured by thromboelastography (TEG), for clinically significant hyperfibrinolysis have not been compared across the 2 US Food and Drug Administration–approved instruments (the TEG 5000 and TEG 6s [Haemonetics]).

Methods

We performed a retrospective, single-center analysis of these 2 instruments using the kaolin (CK) reagent.

Results

Local verification studies showed that the TEG 5000 and TEG 6s CK LY30 upper limits of normal (ULNs) were distinct (5.0% and 3.2%, respectively). Retrospective analysis of patient data showed that abnormal LY30 was 6 times more prevalent with the TEG 6s than with the TEG 5000 instrument. LY30 was a significant predictor of mortality with both instruments (TEG 6s: receiver operating characteristic [ROC] area under the curve [AUC] = 0.836, P ≤ .0001; TEG 5000: ROC AUC = 0.779, P = .028). The optimal LY30 cut point was determined based on these mortality data for each instrument. The TEG 6s showed superior mortality prediction than the TEG 5000 at lower LY30 levels (≥10%), with likelihood ratios of 8.22 and 2.62 for the TEG 6s and TEG 5000, respectively. Patients with a TEG 6s CK LY30 of 10% or higher were significantly more likely to die, receive cryoprecipitate, receive transfusions, or receive massive transfusion than patients with a TEG 6s LY30 of 3.3% to 9.9% (all P < .01). Patients with a TEG 5000 LY30 of 17.1% or higher were significantly more likely to die or use cryoprecipitate (P < .05); transfusion and massive transfusion protocol were not significantly different. Whole blood spiking studies showed that 70 ng/mL tissue plasminogen activator (tPA) achieved an average LY30 of approximately 10% for both instruments.

Conclusions

CK LY30 above the ULN is a sensitive but not specific cutoff for hyperfibrinolysis. At least moderately elevated CK LY30 carries more clinical portent on the TEG 6s instrument than on the TEG 5000. These TEG instruments are not sensitive to low concentrations of tPA.
Significance of concurrent HPV testing with unsatisfactory Papanicolaou test for prediction of follow-up HPV, Papanicolaou test, and biopsy results
Authors: Connelly Courtney F , Collins Nikosa , Cimic Adela , Baskota Swikrity U
Published: July 2023

Objectives

Approximately 1% to 2% of routine cytologic specimens collected for Papanicolaou testing are unsatisfactory for evaluation. The American Society for Colposcopy and Cervical Pathology 2019 guidelines recommend repeat testing within 2 to 4 months of an unsatisfactory Papanicolaou test (UPT) result.

Methods

We evaluated the utility of follow-up Papanicolaou testing, human papillomavirus (HPV) testing, and biopsy in 258 cases of UPTs.

Results

High-risk HPV testing was positive in 17.4% (n = 45) and negative in 82.6% (n = 213) of cases at the time of initial UPT; 8.1% (n = 21) of cases had discordant HPV test results. Similarly, 3.8% (n = 8) of initially HPV-negative cases were reported to be HPV-positive on follow-up; 28.9% (n = 13) of initially HPV-positive cases were reported to be HPV negative on follow-up. In total, 27.1% (n = 70) of cases underwent biopsy. Biopsies with significant findings were present in 40% (n = 12) of HPV-positive cases and 7.5% (n = 3) of HPV-negative cases. Low-grade squamous intraepithelial lesion (LSIL) (low-grade cervical intraepithelial neoplasia [CIN-1]) was the most significant finding in all 3 HPV-negative biopsies; 58.3% (n = 7) of HPV-positive biopsies showed LSIL (CIN-1), 13.3% (n = 4) showed HSIL (high-grade CIN), and 3.3% (n = 1) showed invasive carcinoma. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of concurrent HPV testing at the time of UPT for predicting follow-up HPV test result within 1 year of initial UPT are 80.0%, 94.0%, 71.1%, and 96.2%, respectively. The sensitivity, specificity, PPV, and NPV of initial HPV test results for predicting follow-up Papanicolaou test results are 67.7%, 89.7%, 48.8%, and 95.0%, respectively.

Results

Concurrent HPV testing in the setting of UPT can be a sensitive tool for predicting follow-up HPV status and significant findings of squamous intraepithelial lesions on follow-up Papanicolaou tests and biopsy.
Short Course Training on a Quality Management System for Pathologists, Trainees, and Histotechnologists During the African Pathology Assembly
Authors: Guarner Jeannette , Hale Martin John , Milner Danny A , Nelson Ann Marie
Published: July 2023

Objectives

Provide quality management training in anatomic pathology so that slides are of adequate quality and can be interpreted.

Methods

During the first African Pathology Assembly, we performed a needs assessment and knowledge quizzes, then presented 4 modules of the quality management system (personnel management, process control, sample management, and equipment) that are used to train quality in vertical programs by the World Health Organization.

Results

Participants included 14 (34%) trainees, 14 (34%) pathologists, and 9 (22%) technologists from South Africa (11), Nigeria (6), Tanzania (4), and other countries (18). Thirty (73%) participants took the course because they had interest in the topic while 6 (15%) did it because it was recommended by a supervisor. Most participants thought that the quality of slides was medium to high in their institution and that clinicians trust results. The most frequent quality issues cited included problems from processing to staining, long turnaround times, and preanalytical issues (fixation, lack of clinical history). The average result of the knowledge quiz was 6.7 (range, 2-10) before (38 participants) the course and 8.3 (range, 5-10) after (30 participants) the course.

Conclusions

This assessment suggests there is a need for quality management courses in pathology in Africa.
The role of within-plant variation in nectar production: an experimental approach
Authors: Maldonado Michelle , Fornoni Juan , Boege Karina , Pérez Ishiwara Rubén , Santos-Gally Rocío , Domínguez César A
Published: July 2023

Background and aims

Nectar, a plant reward for pollinators, can be energetically expensive. Hence, a higher investment in nectar production can lead to reduced allocation to other vital functions and/or increased geitonogamous pollination. One possible strategy employed by plants to reduce these costs is to offer variable amounts of nectar among flowers within a plant, to manipulate pollinator behaviour. Using artificial flowers, we tested this hypothesis by examining how pollinator visitation responds to inter- and intra-plant variation in nectar production, assessing how these responses impact the energetic cost per visit.

Methods

We conducted a 2 × 2 factorial experiment using artificial flowers, with two levels of nectar investment (high and low sugar concentration) and two degrees of intra-plant variation in nectar concentration (coefficient of variation 0 and 20 %). The experimental plants were exposed to visits (number and type) from a captive Bombus impatiens colony, and we recorded the total visitation rate, distinguishing geitonogamous from exogamous visits. Additionally, we calculated two estimators of the energetic cost per visit and examined whether flowers with higher nectar concentrations (richer flowers) attracted more bumblebees.

Key Results

Plants in the variable nectar production treatment (coefficient of variation 20 %) had a greater proportion of flowers visited by pollinators, with higher rates of total, geitonogamous and exogamous visitation, compared with plants with invariable nectar production. When assuming no nectar reabsorption, variable plants incurred a lower cost per visit compared with invariable plants. Moreover, highly rewarding flowers on variable plants had higher rates of pollination visits compared with flowers with few rewards.

Conclusions

Intra-plant variation in nectar concentration can represent a mechanism for pollinator manipulation, enabling plants to decrease the energetic costs of the interaction while still ensuring consistent pollinator visitation. However, our findings did not provide support for the hypothesis that intra-plant variation in nectar concentration acts as a mechanism to avoid geitonogamy. Additionally, our results confirmed the hypothesis that increased visitation to variable plants is dependent on the presence of flowers with nectar concentration above the mean.
Understanding Platysma Muscle Contraction Pattern and Its Relationship to Platysmal Banding: A Real-Time Ultrasound Study
Authors: Calomeni Mariana , Alfertshofer Michael , Moellhoff Nicholas , Freytag David L , Nikolis Andreas , Biesman Brian , Davidovic Kristina , Schelke Leonie , Velthuis Peter J , Cotofana Sebastian
Published: July 2023

Background

Addressing neck contouring with surgical and nonsurgical aesthetic procedures includes understanding the origin of platysmal banding. A theory was postulated to explain this phenomenon by isometric vs isotonic muscular contraction patterns. However, no scientific proof had been provided to date for its correctness.

Objectives

The aim of this study was to confirm the correctness of the platysmal banding theory based on isometric vs isotonic muscular contractions.

Methods

Eighty platysma muscles from 40 volunteers (15 males and 25 females) were investigated (mean age 41.8; SD 15.2 years; mean BMI of 22.2; SD 2.3 kg/m2). Real-time ultrasound imaging was utilized to measure the increase in local muscle thickness inside and outside of a platysmal band as well as platysma mobility.

Results

Within a platysmal band, the local thickness of the muscle increases during muscular contractions by 0.33 mm (37.9%; P < .001). Outside of a platysmal band the thickness of the platysma muscle decreased by 0.13 mm (20.3%; P < .001). It was identified that within a platysmal band no gliding was detectable, whereas outside of a band an average muscle gliding of 2.76 mm was observed.

Conclusions

The results confirm the correctness of the isometric vs isotonic platysma muscle contraction pattern theory: isotonic muscle contraction (gliding without increase in tension and therefore in muscle thickness) vs isometric muscle contraction (no gliding but increase in tension and therefore in muscle thickness). These 2 types of contraction patterns occur within the platysma simultaneously and are an indicator for zones of adhesion in the neck to guide surgical and nonsurgical aesthetic procedures.

Level of Evidence: 4
EnGens: a computational framework for generation and analysis of representative protein conformational ensembles
Authors: Conev Anja , Rigo Mauricio Menegatti , Devaurs Didier , Fonseca André Faustino , Kalavadwala Hussain , de Freitas Martiela Vaz , Clementi Cecilia , Zanatta Geancarlo , Antunes Dinler Amaral , Kavraki Lydia E
Published: July 2023

Proteins are dynamic macromolecules that perform vital functions in cells. A protein structure determines its function, but this structure is not static, as proteins change their conformation to achieve various functions. Understanding the conformational landscapes of proteins is essential to understand their mechanism of action. Sets of carefully chosen conformations can summarize such complex landscapes and provide better insights into protein function than single conformations. We refer to these sets as representative conformational ensembles. Recent advances in computational methods have led to an increase in the number of available structural datasets spanning conformational landscapes. However, extracting representative conformational ensembles from such datasets is not an easy task and many methods have been developed to tackle it. Our new approach, EnGens (short for ensemble generation), collects these methods into a unified framework for generating and analyzing representative protein conformational ensembles. In this work, we: (1) provide an overview of existing methods and tools for representative protein structural ensemble generation and analysis; (2) unify existing approaches in an open-source Python package, and a portable Docker image, providing interactive visualizations within a Jupyter Notebook pipeline; (3) test our pipeline on a few canonical examples from the literature. Representative ensembles produced by EnGens can be used for many downstream tasks such as protein–ligand ensemble docking, Markov state modeling of protein dynamics and analysis of the effect of single-point mutations.
OralsO01 Loss of TGFBR2 and activation of PI3 kinase signalling is associated with metastatic cutaneous squamous cell carcinomas
Authors: Treanor-Taylor Mairi , Zhang Ying , Schreyer Daniel  , Harwood Catherine , Leigh Irene  , Bailey Peter , Wotton David , Inman Gareth
Published: July 2023

Cutaneous squamous cell carcinoma (cSCC) can be a relatively low-risk cancer type as it is typically curable upon surgical resection; however, in advanced cases, treatment options are lacking and patient outcomes are poor. The molecular mechanisms that drive metastasis and the associated targets for therapeutic intervention are yet to be determined. To investigate drivers of metastatic cSCC, we undertook whole-genome sequencing of matched primary and metastatic cSCC samples from two patients. Analysis of single nucleotide variants and copy number changes revealed the high tumour mutational burden of this cancer type due to ultraviolet-induced DNA damage, highlighting the challenge faced in filtering out passenger mutations and identifying oncogenic events. Variations unique to the metastases were obtained, and their pathogenicity was assessed. Damaging mutations were found in transforming growth factor beta receptor 2 (TGFBR2) and genes in the PTEN/PI3K/Akt signalling pathway, specifically PTEN, PIK3R1 and PIK3CA, which have previously occurred in cancer samples included in COSMIC, cBioPortal, Cancer Hotspots and Genomic Data Commons. To validate functionally the role of TGFBR2 and PTEN/PI3K/Akt pathway mutations in skin cancers, we generated conditional, inducible Ptenfl/fl;K14Cre+, Pten+/fl;Tgfbr2fl/fl;K14Cre+ and Ptenfl/fl;Tgfbr2fl/fl; K14Cre+ mice. Loss of both Tgfbr2 and Pten in mice was associated with increased proliferation (P = 2.1 × 106), invasion, lymph node metastases and poorer tumour-free (P < 9.0 × 107) and overall survival (P < 2.0 × 106). These data showcase the role of transforming growth factor-β and PTEN/PI3K/Akt signalling pathways on the development of metastatic cSCC.
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

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 < 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

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

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

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

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

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

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

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

Objective

To map inflammatory biomarkers in patients with autonomous cortisol secretion (ACS) and overt Cushing syndrome (CS).

Method

Observational 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).

Results

Combined, 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.

Conclusion

There 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

Background

Diminished heart rate variability (HRV) has been observed in epilepsy, especially in epilepsy with depressive disorders. However, the underlying mechanism remains elusive.

Methods

We 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.

Results

We 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.

Conclusions

Our 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.
T-Cell Immunity Against Severe Acute Respiratory Syndrome Coronavirus 2 Measured by an Interferon-γ Release Assay Is Strongly Associated With Patient Outcomes in Vaccinated Persons Hospitalized With Delta or Omicron Variants
Authors: Fernández-González Marta , Agulló Vanesa , García José Alberto , Padilla Sergio , García-Abellán Javier , de la Rica Alba , Mascarell Paula , Masiá Mar , Gutiérrez Félix
Published: July 2023

Background

We measured T-cell and antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vaccinated patients hospitalized for coronavirus disease 2019 (COVID-19) and explored their potential value to predict outcomes.

Methods

This was a prospective, longitudinal study including vaccinated patients hospitalized with Delta and Omicron SARS-CoV-2 variants. TrimericS-IgG antibodies and SARS-CoV-2 T-cell response were measured using a specific quantitative interferon-γ release assay (IGRA). Primary outcome was all-cause 28-day mortality or need for intensive care unit (ICU) admission. Cox models were used to assess associations with outcomes.

Results

Of 181 individuals, 158 (87.3%) had detectable SARS-CoV-2 antibodies, 92 (50.8%) showed SARS-CoV-2–specific T-cell responses, and 87 (48.1%) had both responses. Patients who died within 28 days or were admitted to ICU were less likely to have both unspecific and specific T-cell responses in IGRA. In adjusted analyses (adjusted hazard ratio [95% confidence interval]), for the entire cohort, having both T-cell and antibody responses at admission (0.16 [.05–.58]) and Omicron variant (0.38 [.17–.87]) reduced the hazard of 28-day mortality or ICU admission, whereas higher Charlson comorbidity index score (1.27 [1.07–1.51]) and lower oxygen saturation to fraction of inspired oxygen ratio (2.36 [1.51–3.67]) increased the risk.

Conclusions

Preexisting immunity against SARS-CoV-2 is strongly associated with patient outcomes in vaccinated individuals requiring hospital admission for COVID-19. Persons showing both T-cell and antibody responses have the lowest risk of severe outcomes.
Effect of China’s long-term care insurance on health outcomes of older disabled people: role of institutional care
Authors: Zhou Jin-Qiu , Zhu Bin , Yan Yu-Hua , Hou Li-Sha , Wu Jin-Hui , Dong Bi-Rong , Dou Qing-Yu
Published: July 2023

Since the public long-term care insurance (LTCI) system was piloted in Chengdu, China, in October 2017, there has been considerable growth of LTC institutions in China. This study aimed to evaluate the health value effect of LTCI in older patients with severe disabilities in an LTC institution. This prospective study was based on data from 985 severe disability patients with or without LTCI from October 2017 to May 2021 in the Eighth People’s Hospital, Chengdu, China. The Cox proportional hazard model estimated LTCI’s health value, including survival probability and risk of pneumonia/pressure ulcers. Subgroup analysis was performed for sex, age, Charlson Comorbidity Index (CCI), and the number of drugs. In the analysis, 519 and 466 patients in LTCI and non-LTCI groups were included, respectively. In adjusted Cox analyses, the LTCI group had a significantly elevated survival rate compared with the non-LTCI groups at 12 months (P < .001, hazard ratio (HR) = 1.758, 95% confidence interval (CI) 1.300–2.376). At 40 months, the adjusted survival rate was 62.6% in the LTCI group, which was significantly higher (53.7%; P = .003, HR = 1.438, 95% CI 1.131–1.831). The subgroups of patients aged 60 to 79 years (interaction P = .007) and with CCI ≥ 3 (interaction P = .026) were more significantly associated with survival improvement than those aged >80 years and with CCI< 3. The LTCI group was also at lower risk for hospital-acquired pneumonia (P = .016, HR 0.622, 95% CI 0.422–0.917) and pressure ulcers (P = .008, HR 0.695, 95% CI 0.376–0.862). The improved survival of LTCI remained stable in sensitivity analyses. For older patients with severe disabilities, in a LTC institution, LTCI significantly improved their health profile and longevity after a year, suggesting the large role and development potentiality of institution care in the LTCI system of China.
Viral etiology of acute respiratory tract infection among children under 5 years of age in Kunming City, China: a matched case–case–control study
Authors: Li Ming , Li Cuilian , Jian Xiaoli , Han Dingrui , Zhao Jinglin , Jiang Li
Published: July 2023

Aims

Nucleic acid-based molecular techniques in current laboratory practice allow the identification of a broad range of respiratory viruses. However, due to asymptomatic carriage, the detection of viruses in the respiratory tract does not necessarily indicate disease. The study aimed to investigate infections of different viruses that colonize the airways, the viral combinations in coinfection, and the viral association with the occurrence of either upper respiratory tract infection (AURTI) or lower respiratory tract infection (ALRTI) in children.

Methods and results

A matched case–case–control study included ALRTI cases, AURTI cases, and healthy controls was conducted at Kunming Children’s Hospital. Oropharyngeal swabs from the three groups were collected for eight viral pathogens detection by multiplex RT-PCR. The association of each pathogen with disease status was determined by comparing the results between cases and controls. From 1 March 2021 through 28 February 2022, 278 participants in each group were investigated. Viral infection was detected in 54.0%, 37.1%, and 12.2% of the ALRTI cases, AURTI cases, and healthy controls, respectively. Human respiratory syncytial virus (RSV), adenovirus (ADV), and parainfluenza virus-3 (PIV-3) were the most frequently documented viruses. RSV/ADV was the most frequent combination detected in coinfection. When compared to healthy controls, RSV and PIV-3 were independently associated with both ALRTI and AURTI.

Conclusions

RSV and PIV-3 were causes of both ALRTI and AURTI cases. These results provide initial evidence of the potential of microbiota-based diagnostics for the differential diagnosis of severe acute respiratory infections using oropharyngeal swab samples.
A lifecycle framework illustrates eight stages necessary for realizing the benefits of patient-centered clinical decision support
Authors: Sittig Dean F , Boxwala Aziz , Wright Adam , Zott Courtney , Desai Priyanka , Dhopeshwarkar Rina , Swiger James , Lomotan Edwin A , Dobes Angela , Dullabh Prashila
Published: July 2023

The design, development, implementation, use, and evaluation of high-quality, patient-centered clinical decision support (PC CDS) is necessary if we are to achieve the quintuple aim in healthcare. We developed a PC CDS lifecycle framework to promote a common understanding and language for communication among researchers, patients, clinicians, and policymakers. The framework puts the patient, and/or their caregiver at the center and illustrates how they are involved in all the following stages: Computable Clinical Knowledge, Patient-specific Inference, Information Delivery, Clinical Decision, Patient Behaviors, Health Outcomes, Aggregate Data, and patient-centered outcomes research (PCOR) Evidence. Using this idealized framework reminds key stakeholders that developing, deploying, and evaluating PC-CDS is a complex, sociotechnical challenge that requires consideration of all 8 stages. In addition, we need to ensure that patients, their caregivers, and the clinicians caring for them are explicitly involved at each stage to help us achieve the quintuple aim.