bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of Hypoxic Condition on Cytotoxicity, Cellular Migration, and Osteogenic Differentiation Potential of Aged Periodontal Ligament Cells
Authors: Jaiklaew Sukrit , Tansriratanawong Kallapat 
Published: May 2024

Abstract
Objective This study aimed to investigate and compare the influence of hypoxic conditions on cytotoxicity, cellular migration, and osteogenic differentiation of aged periodontal ligament (PDL) cells.
Materials and Methods Isolated human PDL cells from aged and young subjects were cultured under hypoxic conditions, which were treated with hydrogen peroxide (H2O2) (0, 25, 50, 100, 200, and 500 µM). To assess cytotoxicity, lactate dehydrogenase release was determined by the optical density at 490 nm, and the percentage of cell death was calculated. An in vitro wound healing assay was performed over 24 to 48 hours for cellular migration determination. Osteogenic differentiation was determined by alizarin red staining and osteogenic gene expression, including the expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN).
Results There was a significant difference in the percentage of cell death with high hypoxic condition (200 and 500 µM) compared to low hypoxic conditions on both day 1 and 2. The highest cellular migration was depicted at 50 µM in both young and aged groups of the in vitro wound healing assay. Osteogenic gene expression of RUNX2 in the aged group was increased at 25 and 50 µM hypoxic condition at day 7, but the expression was gradually decreased after 14 days. On the contrary, the expression of ALP and OPN in the aged group was increased at day 14. Only OPN had been found to be statistically significantly different when compared with gene expression at day 7 and 14 (p < 0.05). The results showed no statistically significant differences when compared with the young and aged groups in all genes and all concentrations.
Conclusion The concentration of low hypoxic condition (25–50 µM) was proposed to promote cell viability, cellular migration, and osteogenic differentiation in aged PDL cells. We suggested that the potential of aged PDL cells for use in cell therapy for periodontal regeneration might possibly be similar to that of young PDL cells.
Assessing the status quo of international employees’ adjustment research, 1990–2022: a review and future research agenda
Authors: Baneviciene Irma , Andresen Maike , Kumpikaite-Valiuniene Vilmante 
Published: May 2024

AbstractThis study aims to comprehensively review scientific journal articles related to the adjustment of international employees within the management and business domain from 1990 to 2022. The study seeks to identify trends and patterns in research topics and to propose a future research agenda. To achieve this, we analysed 222 articles from the Web of Science Core Collection database through two main steps: (1) a bibliometric analysis to track the field’s evolution over time and (2) a content analysis of abstracts to examine covered topics and pinpoint research gaps. Our findings indicate that the theory surrounding the adjustment of international employees is still in the process of maturation, with several potential areas for future research emerging. The analysis reveals that factors influencing adjustment are the most extensively researched for assigned expatriates, leaving other international employees relatively under-researched. Moreover, quantitative research emerged as the most prevalent methodological approach among the included studies. Most study samples predominantly consisted of individuals moving between Asia, Europe, and North America, underscoring the significance of Africa—characterised by substantial migration flows within the region—as a focal point for future adjustment research. Moreover, individual-, organisation-, and country-related antecedents of international employees’ anticipatory and in-country adjustments were analysed to present conclusions for future research. This study supplements the domains of international human resource management and international business by identifying research priorities concerning the adjustment of international employees and outlining an agenda for further research.
Polypyrrole film formation using DC biasing of substrate in in-solution plasma process
Authors: Jang Hyojun , Jung Eun Young , Kim Jae Young , Tae Heung-Sik 
Published: May 2024

Beyond the existing in-solution plasma (iSP) process used for nanoparticle synthesis, polymer film synthesis has recently been demonstrated via iSP, which occurs at a specific cycle in a substrate with electrical potential. Herein, we propose an iSP reactor designed to tune the characteristics of polypyrrole (PPy) films by applying varied DC biases to the substrate during the solution plasma process. Applying DC bias to the substrate notably enhances the iSP during the negative cycle, thereby generating ionic precursors from pyrrole monomers for PPy film formation. These films demonstrated morphological variances as a function of the applied VDC. At a low voltage (VDC = 0.5 kV), a uniform film was formed via a layer-by-layer growth mechanism, and as the voltage was increased, the film exhibited a structured morphology. Specifically, at VDC = 0.5 kV, the PPy film was successfully coated onto a finely patterned electrode. The proposed iSP process, under specific DC biasing conditions, enables polymer coating without necessitating additional chemicals, catalysts, or masks. This method holds potential for a wide range of applications.
Situationally-triggered metaphor as political argument
Authors: Augé Anaïs 
Published: May 2024

Abstract
This paper proposes to investigate the public responses to situationally-triggered metaphors as these have been
observed in political argumentation. Situationally-triggered metaphors occur when a nonmetaphorical connection is made between the
metaphor and an aspect of the relevant situational context. The question addressed in this research is: how are such metaphors
perceived by the public when these form part of the political argumentation? To answer this question, the study focuses on a
particular instance of political situationally-triggered metaphor i.e., Boris Johnson’s “James Bond” metaphor produced during
COP26. The paper draws on Critical Metaphor Analysis and Deliberate Metaphor Theory to analyse the public comments and reactions
posted on the social media platform Twitter in response to the politician’s arguments. The analysis reveals that most of the
public responses exploit the “James Bond” metaphor to dispute Johnson’s self-identification to the fictional character and provide
meta-arguments that revolve around the politician’s misuse of metaphors. In contrast, responses that exploit the metaphor to
convey political arguments or endorsement are much more limited. It is thus argued that situationally-triggered metaphors not only
represent a political rhetorical device, but they are also effective political tools to shift public attention towards discursive
patterns instead of arguments presented in discourse.
Photon counting based pump-probe technique for quantitative characterization of fluorescence in a lock-in free detection manner
Authors: Rehman Khalil Ur , Das Subir , Kao Fu-Jen 
Published: May 2024

Abstract
The stimulated emission (SE) signal in pump-probe experiment is conventionally measured with lock-in detection to differentiate the weak signals from the relatively large background of spontaneous emission and probe beam. Therefore, direct characterization of signal strength are often major limiting factors in terms of noise, speed, and data acquisition. In contrast, photon counting allows direct quantification of signal strength, while synchronized pump-probe pulse enables precise timing and the separation of signals accordingly. Herein, the SE based pump-probe method is combined with time-correlated single-photon counting to investigate the ultrafast photochemical parameters, digitally and quantitatively. As a proof-of-concept, our technique is applied to investigate, fluorescence lifetime

(
τ
)

∼
3.71

ns

, optical absorption cross-section

(

σ

abs

)

∼
1.23
×

10

−
16

c

m

2

, and the SE cross-section

(

σ

SE

)

∼
2.22
×

10

−
17

c

m

2

, of a fluorescent dye (ATTO 647N) quantitatively. The experimental results are also compared with theoretical photon statistics to further justify the advantages including experimental and statistical critical molecular dynamics parameters extraction with excellent high accuracy.
O16 Understanding the pleiotropic effects of the Rho-kinase inhibitor Y-27632 on human keratinocytes
Authors: Flynn Benjamin , Glallacher Kelli , Walko Gernot 
Published: May 2024

Abstract

Introduction and aims
Successful epidermal transplant generation depends on sufficiently high numbers of self-renewing stem cells that give rise to clonal colonies termed ‘holoclones’. Treatment with Y-27632, a nonspecific Rho-kinase 1/2 inhibitor, conditionally immortalizes epidermal keratinocyte cultures. However, the pleiotropic signalling mechanisms downstream of Y-27632 remain largely unexplored. We have therefore characterized the effects of continuous long-term Y-27632 treatment on chromatin regulation, and investigated whether Y-27632 reprogrammes a holoclone-like state.

Methods
Neonatal foreskin-derived human epidermal keratinocytes were cultured from passage p5 to p30, with or without Y treatment. Samples were collected for RNA sequencing analysis from progenitor-enriched middle passage cells (p5, MPK), progenitor-depleted late-passage cells (p8, LPK) and Y-treated cells at p8 (LPK-Y). Samples were also collected for assay for transposase-accessible chromatin (ATAC)-Seq analysis to assess open and closed chromatin regions.

Results
Untreated cultures became depleted of proliferating cells by p11, whereas Y maintained a highly proliferative culture up to p30 and beyond. Y withdrawal at p11 resulted in rapid loss of proliferative capacity within a single passage. ATAC-Seq identified 196 differentially regulated chromatin sites in MPK vs. LPK. Y treatment maintained accessibility in approximately 40% of these MPK-enriched chromatin regions. Approximately 3000 differentially expressed genes (DEGs) were identified in MPK vs. LPK, while Y treatment induced approximately 11 000 DEGs. Activated genes in LPK-Y were enriched for a holoclone signature, with no evidence for activation of a precancerous transcriptome. Y treatment altered the expression of various genes associated with amino acid metabolism, enabling cells to overcome serine/glycine autotrophy.

Conclusions
Y acts by preserving accessibility of progenitor-enriched chromatin sites and further reprogrammes the keratinocyte transcriptome towards a more holoclone-like state. However, keratinocytes retain a ‘memory’ of their doubling number, which is conditionally suppressed by Y. The observed global transcriptomic changes are indicative of the highly pleiotropic nature of the Y-induced signalling changes, warranting further deconvolution.
O17 Highly efficient base editing of homozygous and heterozygous COL7A1 variants in fibroblasts from patients with recessive dystrophic epidermolysis bullosa using adenine base editor 8e mRNA system delivered via novel lipid nanoparticle formulations
Authors: Guri Ina , Brooks Imogen , Alrokh Yara , Kazamizadeh Aidni , Graham Carina , Hart Steven , McGrath John , Jackow Joanna 
Published: May 2024

Abstract

Introduction and aims
Adenine base editors (ABEs) present an attractive approach to gene editing in the inherited blistering disease recessive dystrophic epidermolysis bullosa (RDEB), which Results from pathogenic variants in COL7A1. We previously showed that ABE8e, a new ABE variant, confers remarkable efficiency (94.6%) and a favourable safety profile in correcting variants and restoring collagen 7 (C7) expression when electroporated into RDEB fibroblasts. With a goal to develop topical base editing creams, we now explore use of lipid nanoparticle (LNP)-encapsulated ABE8e mRNA system to edit two distinct variants in RDEB fibroblasts.

Methods
Primary patient fibroblasts harbouring a homozygous G&gt;A mutation (exon 42) or a heterozygous C&gt;T mutation (exon 54) were gene edited using ABE8e mRNA, delivered via lipofectamine or six different LNPs. To assess the gene editing efficiency, Sanger sequencing and next-generation sequencing were employed. Cell viability was evaluated via a lactate dehydrogenase cytotoxicity assay, while functional restoration of C7 expression was assessed via Western blotting of cell lysate and supernatant. Finally, topical delivery was tested on normal human skin equivalents (HSEs).

Results
We identified an optimal ratio of 1 : 7 for sgRNA : ABE8e, at which editing efficiency increases with ABE8e dosage, with 400 ng being sufficient for 90% biallelic editing. Formulations containing DTDTMA (C14) and DHDTMA (C16) LNPs demonstrated approximately 90% and approximately 80% editing (with and without the peptide, respectively), in addition to reduced cytotoxicity compared with lipofectamine. Depending on the nature of the mutation, the edit corresponded to an increase in C7 expression in supernatant only or cell lysate as well, whereby C14 LNPs enabled twofold C7 restoration. Finally, our HSEs demonstrated viability of topical delivery of lipofectamine-encapsulated green fluorescent protein mRNA.

Conclusions
We demonstrated the potential value of base editing as a gene therapy for RDEB in addition to the potential of these LNPs for clinical translation of the therapy. Nonetheless, further safety and functionality tests still need to be conducted.
P09 Next-generation sequencing: on-and off-target analysis of gene editing in fibroblasts from patients with recessive dystrophic epidermolysis bullosa
Authors: Kazemizadeh Aidin , Brooks Imogen , Guri Ina , Alrokh Yara , Balon Katarzyna , Laczmanski Lukasz , McGrath John , Jackow-Malinowska Joanna 
Published: May 2024

Abstract

Introduction and aims
Base editors (BEs) and prime editors (PEs) have proved to have advantages over traditional clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 in correcting pathogenic mutations. We previously demonstrated 94.6% efficiency using adenine base editor (ABE)8e in correcting nonsense mutation (c.5047 C&gt;T) in fibroblasts, which causes recessive dystrophic epidermolysis bullosa (RDEB). However, safety profile and cell-type-specific efficiencies of gene editing techniques have not been characterized. The aim of our research is to establish corrections of c.5047 C&gt;T pathogenic variant in fibroblasts and compare off-target effects and efficiency of CRISPR-Cas9, BE and PE systems.

Methods
Primary fibroblasts harbouring heterozygous C&gt;T pathogenic variant on exon 54 will be transfected with CRISPR-Cas9, ABE8e and PE using electroporation or lipofectamine. Both Sanger sequencing and next-generation sequencing (NGS) will be conducted to confirm pathogenic variant corrections. Galaxy platform which incorporates different NGS analysis tools will be used to analyse the NGS data. Subsequently, whole-exome sequencing and targeted amplicon sequencing will be used for off-target analysis.

Results
On-target efficiency for ABE8e to correct c.5047 C&gt;T pathogenic variant in patient primary fibroblasts was determined first by Sanger sequencing showing 100% editing in contrast to 84% determined by NGS. Moving forward, we plan to correct the same pathogenic variant with PEs. Initially experiments are under way with PEmax mRNA and engineered PE guide RNAs (epegRNA) to compare different PEs. Furthermore, we will investigate the off-target effects of CRISPR-Cas9, ABE8e and PEs to edit RDEB primary fibroblasts using NGS Methods and conduct functional studies.

Conclusions
Here, we showed that the c.5047 C&gt;T pathogenic variant in RDEB fibroblasts could be successfully edited using ABE8e. We demonstrated that NGS is required to precisely determine the editing efficacy. Taken together, we have established a pipeline for base editing in RDEB and we aim to achieve the same for prime editing in the future.
P15 Understanding the role of cathepsin H in atopic dermatitis pathogenesis
Authors: Tachie-Menson Theresa , Hughes Aaron , O’Toole Edel , O’Shaughnessy Ryan 
Published: May 2024

Abstract

Introduction and aims
Atopic dermatitis (AD) is a chronic skin condition ranked the 15th highest global burden for all nonfatal diseases. Our lab has found that patients with AD have a reduction in the gene and protein expression of cathepsin H, a lysosomal cysteine protease with aminopeptidase activity. Ctsh-/- mice have a reduced integrity of the epidermal barrier, including reduced filaggrin processing, reduced corneocyte integrity and a change in the expression of several proinflammatory mediators including interleukin-1α. We aim to elucidate the roles of cathepsin H in keratinocytes to understand its contribution to the pathogenesis of AD.

Methods
We utilized rat epidermal and N/TERT keratinocytes transiently overexpressing procathepsin B, H or L in addition to incubating keratinocytes with cathepsin H inhibitor (CTSHi) for 24 h to identify potential substrates and proteins upregulated or downregulated with cathepsin H expression. We also performed colocalization studies using indirect immunofluorescence in rat epidermal keratinocytes (REKs) and N/TERTs to investigate the biology of cathepsin H.

Results
REKs transiently overexpressing procathepsin H-FLAG or FLAG and REKs treated with the cathepsin H inhibitor H2N-Ser(OBzl)-CHN2 were analysed by liquid chromatography-mass spectrometry. This analysis indicated that keratin 1 and keratin 9 were upregulated with cathepsin H expression and downregulated by cathepsin H inhibition. Overexpressed procathepsin H and endogenous cathepsin H do not colocalize with the lysosomal membrane protein, LAMP1, whereas endogenous cathepsin B does.

Conclusions
Keratin 1 and keratin 9 could be coregulated with cathepsin H. Cathepsin H may not primarily localize to lysosomes unlike cathepsin B.
P16 Fibroblast signalling crosstalk drives junctional epidermolysis bullosa disease progression
Authors: Khan Mohammad R A , Kirk Thomas , Caley Matthew P , Rognoni Emanuel 
Published: May 2024

Abstract

Introduction and aims
Junctional epidermolysis bullosa (JEB) is a devastating hereditary skin disorder characterized by severe fragility of skin and mucus membrane, emerges as blistering with little or no trauma. The major classification of JEB includes JEB intermediate and JEB severe; the latter is the most common subtype, which is the result of loss of function mutations in the skin basement membrane protein laminin-332. Lacking this protein impairs basement membrane adhesion of epidermal keratinocytes and leads to deregulated epidermal–dermal crosstalk. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their specific involvement in skin diseases is largely unknown. Here, we investigate how fibroblast subpopulations, namely papillary and reticular fibroblasts, impact JEB disease progression and skin regeneration.

Methods
Three-dimensional (3D) organotypic (OT) combining healthy (control) and diseased (JEB) keratinocytes cells with distinct fibroblast subpopulations from different anatomical sites were developed and analysed by immunohistochemistry and RNA-Seq.

Results
JEB OTs with papillary fibroblasts showed increased epidermal growth, invagination of basal layer and deregulated differentiation compared with reticular fibroblasts. RNA-Seq analysis revealed altered activity in key signalling including immune, cytokine, gap junction, mitogen-activated protein kinase and remodelling of keratinization and ECM proteins, particularly in JEB OT cultured with papillary fibroblast. JEB OTs and cells also demonstrated variation in cell proliferation and activation of transforming growth factor-β1 and immune cell signalling. Currently, we are targeting identified signalling pathways in our two-dimensional and 3D cell culture models and identifying druggable targets, before testing selected candidates in our JEB mouse disease model for further preclinical validation.

Conclusions
By dissecting the pathological signalling interactions between keratinocytes and distinct fibroblast subpopulations, our goal is to identify new therapeutic target to improve the skin health and regeneration in patients with JEB.
Molecular Dynamics Simulation for the Acidic Compounds Retention Mechanism Study on Octyl-Quaternary Ammonium Mixed-Mode Stationary Phase
Authors: Chang Chaoqun , Jin Xinghua , Bai Hui , Zhang Fan , Chen Lei 
Published: May 2024

Abstract
With the widespread application of mixed-mode chromatography in separation analysis, it is becoming increasingly important to study its retention mechanism. The retention behavior of acidic compounds on mixed-mode octyl-quaternary ammonium (Sil-C8-QA) columns was investigated by computer simulation. Firstly, the benzoic acid homologues were used as the analytes, and the simulation model was constructed by the Materials Studio. Geometric optimization, annealing and molecular dynamics (MD) simulation of these complexes resulted in optimized conformations. The binding energy, mean square displacement (MSD) and torsion angle distribution generated by MD simulation were then analyzed. The results showed that the more negative binding energy, the greater the MSD and the narrower the torsion angle distribution, indicating that the stationary phase behaves with stronger interaction and retention. The retention behavior of five acidic drugs on the Sil-C8-QA column was then successfully explained by simulation. Acidic drugs are more retentive on the mixed-mode column due to the more substantial interaction brought by the reversed-phase/ion-exchange mixed-mode mechanism compared to other single-mode columns. This simulation method is expected to provide ideas for studying the separation mechanism and predicting the retention behavior of more complex samples.
Neutralising the ECT Sunset Clause Inter Se
Authors: Braoudakis Nikos , Craveia Rosanne , Baldon Clémentine 
Published: May 2024

Abstract
Following a wave of announcements from major EU Member States withdrawing or considering withdrawing from the Energy Charter Treaty (ECT), the European Commission decided to recommend a coordinated withdrawal of the EU and its Member States from the ECT. The European Commission also backs the adoption of a ‘subsequent agreement on the interpretation of the ECT’ between the EU and its Member States that would ‘clarify’ that the ECT and its sunset clause do not apply intra-EU. The sunset clause contained in Article 47(3) of the ECT has been described as a major hurdle in States’ attempt to exit the ECT. However, under an international treaty law analysis, strong arguments confirm the availability of an agreement between a subset of ECT Contracting Parties that would exclude the application of the ECT and its sunset clause in their relations inter se—including the envisaged EU agreement—as a suitable and effective alternative to modernisation of the ECT. Not only does this appear as the most desirable outcome in view of the continuing protection of fossil fuels under the modernised ECT, but such an agreement would also be firmly grounded in public international law.