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Intrahepatic bile duct (IHBD) development produces a morphologically heterogeneous network of large "ducts" and small "ductules" by adulthood. IHBD formation is closely linked to developmental specification of biliary epithelial cells (BECs) starting as early as E13.5, but mechanisms regulating differential IHBD morphology remain poorly understood. Here, we show that duct and ductule development has distinct genetic requirements, with Sox9 required to form the developmental precursors to peripheral ductules in adult livers. By optimizing large-volume IHBD imaging, we find that IHBDs emerge as a homogeneous webbed structure by E15.5 and undergo morphological maturation through 2 weeks of age. Developmental knockout of Sox9 leads to decreased postnatal branching morphogenesis, resulting in adult IHBDs with normal ducts but significantly fewer ductules. In the absence of Sox9, BECs fail to mature and exhibit elevated TGF-β signaling and Activin A. Exogenous Activin A is sufficient to induce developmental gene expression and morphological defects in wild-type BEC organoids, while early postnatal inhibition of Activin A in vivo rescues IHBD morphogenesis in the absence of Sox9. Our data demonstrate that proper IHBD architecture relies on inhibition of Activin A by Sox9 to promote ductule morphogenesis, defining regulatory mechanisms underlying morphological heterogeneity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Hepatitis C virus (HCV) infection induces liver inflammation, activating hepatic stellate cells (HSC) and advancing fibrosis. Studies have indicated that SOX9 overexpression is closely linked to HSC activation. The study aims to identify genes associated with SOX9 and search for potential targets for detecting and treating liver fibrosis., Method: The dataset GSE15654, containing 216 biopsy samples from HCV-induced early-stage cirrhosis patients, was obtained from the GEO database. Prognostic genes were identified through differential gene analysis, LASSO, and Cox regression analyses. CIBERSORT analysis quantified infiltration levels across 22 immune cell types. Constructing a prognostic prediction model using screened genes and conducting preliminary validation using qRT PCR and RNA sequencing techniques., Results: Elevated SOX9 expression correlates with unfavorable outcomes in patients with early-stage liver fibrosis induced by HCV. We identified nine SOX9-related prognostic DEGs in our study. ADAMTS2, ARHGEF5, CCT8, ERG, LBH, FRMD6, INMT, and RASGRF2 were considered risk factors in the disease progression, while DHRS4 was considered a protective factor. SOX9 expression showed a positive correlation with mast cell infiltration, whereas ARHGEF5 and FRMD6 expressions were positively associated with M0 macrophage infiltration. Our combined model surpasses the commonly used APRI and FIB4 indicators in predicting patient prognosis. The testing of clinical samples also preliminarily validated our research results., Conclusion: The prognostic model based on nine SOX9-related DEGs provides an effective tool for forecasting the progression and outcomes of liver fibrosis. This study introduces a new strategy for advancing liver fibrosis prediction and treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Wu Mei Wan (WMW) is a traditional Chinese herbal formula with a long-standing history in Chinese medicine, valued for its therapeutic properties. However, its potential anti-cancer effects, especially against colorectal cancer (CRC), have not been fully elucidated., Aim of the Study: This study aims to investigate the effects of WMW on colorectal cancer stemness and to elucidate the underlying molecular mechanisms, focusing on the modulation of Sox9 expression via the JAK2/STAT3 signaling pathway., Materials and Methods: WMW was prepared and analyzed using UPLC-MS to identify their main components. To study the therapeutic effects of WMW, AOM/DSS-induced CRC mouse models were established. A comprehensive suite of experimental techniques, including in vivo imaging, cell culture, transfection, CCK-8 assays, colony formation assays, wound healing assays, cell migration assays, Western blotting, dot blot analysis, RT-qPCR, immunohistochemistry, cell transcriptome sequencing, and gene set enrichment analysis, were utilized to explore the pharmacological effects and mechanisms of WMW., Results: WMW significantly inhibited CRC cell viability, proliferation, invasion, and migration in vitro. Mechanistically, WMW suppressed CRC stemness by downregulating Sox9 expression through the JAK2/STAT3 signaling pathway. Additionally, the regulation of methylation and demethylation mediated by TET1 and DNMT3a expression was directly associated with the JAK2/STAT3 pathway's modulation of Sox9 expression. In vivo, WMW treatment attenuated CRC progression and metastasis with minimal toxicity., Conclusion: These findings suggest that WMW exerts potent anti-CRC stemness effects by regulating Sox9 via the JAK2/STAT3 signaling pathway, underscoring its potential as a promising therapeutic agent for CRC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cellular senescence, traditionally associated with aging and chronic diseases, has recently been identified as a potential facilitator of tissue regeneration via a senescence-associated secretory phenotype (SASP). In rodents, the meniscus is known to regenerate spontaneously from the surrounding synovium, but the mechanism, and especially its relationship to cellular senescence, remains unclear. This study investigated the contribution of cellular senescence to spontaneous repair of the rat meniscus. We created a rat partial medial meniscectomy (pMx) model to evaluate time-course changes in regenerative tissue. Immunohistochemistry revealed marked increases in p16 expression and senescence-associated beta-galactosidase (SA-β-gal) activity in the regenerating tissue at the early phase after pMx surgery. RNA sequencing of regenerating tissues identified the upregulation of genes related to aging, extracellular matrix organization, and cell proliferation. Fluorescence staining identified high expression of SOX9, a master regulator of cartilage/meniscus development, adjacent to p16-positive cells. In vitro investigations of the effect of SASP factors on synovial fibroblasts (SFs) demonstrated that conditioned medium from senescent SFs stimulated the proliferation and chondrogenic differentiation of normal SFs. In vivo histological evaluation to determine whether selective elimination of senescent cells with a senolytic drug (ABT-263) retarded spontaneous repair of meniscus in vivo confirmed that ABT-263 decreased the meniscus score and expression of SOX9, aggrecan, and type 1 collagen. Our findings indicate that transient senescent cell accumulation and SASP in regenerating tissues beneficially contribute to spontaneous repair of the rat meniscus. Further research into the molecular mechanism will provide a novel strategy for meniscus regeneration based on cellular senescence., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Background: Lately SOX2 and SOX9, transcription factors associated with stemness-like phenotypes of cancer cells, have been linked to tumor growth, metastasis, and resistance to therapy., Methods: This study aimed on evaluating the expression of SOX2 and SOX9 in a large cohort of patients with OSCC including primary and recurrent tumors and corresponding lymph node metastases. Semiautomatic digital pathology scoring was used to determine protein expression and survival analysis was performed to evaluate its prognostic significance., Results: We found a significant downregulation of SOX9 from primary disease to lymph node metastases (p < 0.001). SOX9 expression and the subgroup SOX2 low SOX9 high were significantly correlated with worse overall survival (p < 0.05). Additionally, SOX2 low SOX9 high expression pattern was confirmed as independent prognosticator for overall survival., Conclusions: These results indicate the relevant role of SOX2 and SOX9 in patients with OSCC and show the clinical relevance for further investigation on the molecular mechanisms underlying SOX-related gene expression., (© 2024 The Author(s). Head & Neck published by Wiley Periodicals LLC.)

Humans are widely exposed to phthalates, a common chemical plasticizer. Previous cohort studies have revealed that maternal exposure to monobutyl phthalate (MBP), a key metabolite of phthalates, is associated with neurodevelopmental defects. However, the molecular mechanism remains unclear. Here, we demonstrate that maternal exposure to MBP enhances neural stem cell (NSC) differentiation into astrocytes with highly expressed C3 and LCN2 in mouse offspring, resulting in increased synapse phagocytosis and cognitive dysfunction. Mechanistically, we find that MBP exposure activates the IRE1α/XBP1s (spliced XBP1) stress response pathway, which regulates key genes involved in astrocyte differentiation (SOX9 and ATF3) and reactivity (C3 and LCN2). Conditional knockout or pharmacological inhibition of IRE1α markedly inhibits NSC differentiation into astrocytes and astrocyte reactivity, attenuates synapse phagocytosis, and improves cognitive function. This phenotype is further recapitulated in a human brain organoid model. Together, these findings unveil the molecular mechanism underlying the neurodevelopmental deficits caused by a widespread environmental pollutant., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

SOX9 is a crucial transcriptional regulator of cartilage development and homeostasis. Dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic dysplasia, acampomelic campomelic dysplasia, and scoliosis. Yet how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four pathogenic variants of SOX9 identified in a cohort of patients with congenital vertebral malformations. We report a pathogenic missense variant in the transactivation middle (TAM) domain of SOX9 associated with mild skeletal dysplasia and scoliosis. We isolated a Sox9 mutant mouse with an in-frame microdeletion in the TAM domain ( Sox9 Asp272del ), which exhibits skeletal dysplasia including kinked tails, rib cage anomalies, and scoliosis in homozygous mutants. We find that both the human missense and the mouse microdeletion mutations resulted in reduced SOX9 protein stability in cell culture, while Sox9 Asp272del mutant mice show decreased SOX9 expression in the growth plate and annulus fibrosus tissues of the spine. This reduction in SOX9 expression was correlated with the reduction of extracellular matrix components, such as tenascin-X and the Adhesion G-protein coupled receptor ADGRG6. In summary, our work identified and modeled a pathologic variant of SOX9 within the TAM domain and demonstrated its importance for SOX9 protein stability. Our work demonstrates that SOX9 stability is important for the regulation of ADGRG6 expression, which is a known regulator of postnatal spine homeostasis, underscoring the essential role of SOX9 dosage in a spectrum of axial skeleton dysplasia in humans., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Chemical-induced acute lung injury is characterized by impaired epithelial regenerative capacity, leading to acute pulmonary edema. Numerous studies have investigated the therapeutic potential of endogenous stem cells with particular emphasis on alveolar type 2 epithelial (AEC2) cells owing to their involvement in lung cell renewal. Sox9, a transcription factor known for its role in maintaining stem cell properties and guiding cell differentiation, marks a subset of AEC2 cells believed to contribute to epithelial repair. However, the role of Sox9 + AEC2 cells in the distal lung alveolar cells and the potential roles in chemically induced acute lung injury have never been explored., Methods: In this study, we generated Sox9 flox/flox ;Sftpc Cre-ERT2 mice and examined the effects of Sox9 + AEC2 cells on the pathophysiology of epithelial damage during chemical-induced acute lung injury. Subsequently, Sox9-Cre ERT2 Ai9 mice were used for lineage tracing to elucidate the repair mechanisms., Results: Our findings revealed that Sox9 + AEC2 cells endowed with stem cell properties induced cell proliferation during lung injury, predominantly in the damaged alveolar region. This process is accompanied by the regulation of inflammatory responses and orderly differentiation, thereby promoting epithelial regeneration., Conclusion: These results provide compelling in vivo genetic evidence supporting the characterization of Sox9 + AEC2 cells as bona fide lung epithelial stem cells, demonstrating their multipotency and self-renewal capabilities during lung repair and regeneration. The identification of Sox9 + AEC2 cells as crucial contributors to the promotion of epithelial repair underscores their potential as therapeutic targets in chemical-induced acute lung injury., Competing Interests: Declarations. Ethics approval: All animal experiments for this study were in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0. All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of Jinshan Hospital, Fudan University, China (approval number: 2022-JS-008; date of approval: March 1, 2022; title of approved project: “PTTG1 mediates CD34+CD45+ cells to promote repair of phosgene-induced acute lung injury”). Consent for publication: All authors provided consent for publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Skin-derived stem cells (SDSCs) are a subtype of adult stem cells (ASCs) that are widely harvested and exempt from ethical restrictions in clinical applications. These cells possess capabilities for self-renewal, proliferation, and multi-lineage differentiation. Compared to model animals like rats and mice, pigs exhibit greater physiological similarity to humans. Porcine skin has very similar histological and physiological characteristics to human skin. Therefore, porcine skin is becoming increasingly significant as an in vitro model for research. In this study, porcine skin-derived stem cells (pSDSCs) were isolated and cultured in vitro for experiments. The expression of stemness-related gene SOX9 was detected. RNA sequencing (RNA-seq) results found that the mammalian target of rapamycin (mTOR) signaling pathway was significantly enriched in SOX9 + pSDSCs. To investigate the role of the mTOR signaling pathway, we added rapamycin (RAPA), an inhibitor of the mTOR complex 1 (mTORC1), and found that the proliferation rate of SOX9 + pSDSCs decreased significantly during culture. In addition, western blotting (WB) results demonstrated that mTORC1 promoted proliferation by phosphorylating S6 kinase (S6K) and then activating cyclin D1(CCND1) in SOX9 + pSDSCs. These findings provide insights into the mechanisms of adult stem cell proliferation., Competing Interests: Declarations. Conflict of interest: No potential conflict of interest was reported by the author(s)., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In mammals, SOX9/Sox9 expression in embryonic gonads is essential for male gonadal sex determination. Multiple enhancers of Sox9 have been identified, of which the mXYSRa/Enh13 enhancer plays a crucial role in mice. SOX9 and SRY binding sites within the enhancer have been identified as functional. Simultaneous deletion of both sites in mice resulted in male-to-female sex reversal. However, the existence of other critical functional sequences remains unclear. This study identified an additional functional sequence by generating mice with partial deletions in mXYSRa/Enh13. Two nucleotide substitutions within the sequence were sufficient for male-to-female sex reversal. In vivo binding assay by CUT&RUN revealed that GATA4 binds to the sequence. In vitro luciferase assay showed that GATA4 promotes the enhancer activity and the substitution of the sequence reduces the effect. Taken together, the functional sequence in mXYSRa/Enh13 is essential for testis differentiation and requires GATA4 binding., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Pancreatic cancer is a lethal disease with an insidious onset, and little is known about its early molecular events. Here, we found that the sterol regulatory element-binding protein 1 (SREBP1) expression is gradually upregulated during the initiation of pancreatic cancer. Through in vitro 3D culture of pancreatic acinar cells and experiments in LSL-Kras G12D/+ ;Pdx1-Cre (KC) mice, we found that pharmacological inhibition of SREBP1 suppressed pancreatic tumorigenesis. In vitro, either knockdown or pharmacological inhibition of SREBP1 suppressed tumor proliferation but SREBP1 overexpression promoted tumor proliferation. In LSL-Kras G12D/+ ;Trp53 fl/+ ;Pdx1-Cre (KPC) mice, we confirmed the tumor-promoting role of SREBP1 in pancreatic cancer progression. Mechanistically, we revealed SOX9 as a downstream target of SREPB1. SREBP1 inhibition decreased SOX9 expression in both acinar cells and pancreatic cancer cells. Indeed, we identified SREBP1 binding sites in the SOX9 promoter region and reported that SOX9 is transcriptionally regulated by SREBP1. Taken together, our findings demonstrate that SREBP1/SOX9 inhibition suppresses pancreatic cancer initiation and growth, suggesting that SREBP1 could serve as a potential target for cancer screening and treatment., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The oral mucosa undergoes daily insults, and stem cells in the epithelial basal cell layer regenerate gingiva tissue to maintain oral health. The Iroquois Homeobox 1 (IRX1) protein is expressed in the stem cell niches in human/mouse oral epithelium and mesenchyme under homeostasis. We found that Irx1+/- heterozygous (Het) mice have delayed wound closure, delayed morphological changes of regenerated epithelium, and defective keratinocyte proliferation and differentiation during wound healing. RNA-Seq analyses between WT and Irx1+/- mice at 3 days postinjury (dpi) found impaired epithelial migration and decreased keratinocyte-related genes upon injury. IRX1-expressing cells are found in the gingival epithelial basal cell layer, a stem cell niche for gingival maintenance. IRX1-expressing cells are also found in cell niches in the underlying stroma. IRX1 activates SOX9 in the transient amplifying layer to increase cell proliferation, and EGF signaling is activated to induce cell migration. Krt14CreERT lineage tracing experiments reveal defects in the stratification of the Irx1+/- HET mouse oral epithelium. IRX1 is primed at the base of the gingiva in the basal cell layer of the oral epithelium, facilitating rapid and scarless wound healing through activating SOX9 and the EGF signaling pathway.

Osteoarthritis (OA) is a multi-factorial degenerative joint disease with unclear pathogenesis. Conservative treatments, primarily aimed at pain relief, fail to halt disease progression. Metabolic syndrome has recently been implicated in OA pathogenesis, underscoring the need for novel therapeutic strategies. Arctiin (ARC), a lignan known for its anti-inflammatory and anti-osteoporotic properties, has potential effects on OA that merit exploration. We assessed ARC's impact on chondrocyte viability using the Cell Counting Kit-8 and toluidine blue staining for glycosaminoglycan presence. Gene and protein expression were analyzed via RT-PCR, Western blotting, and immunofluorescence. An OA rat model was employed for in vivo evaluations through histological assessments and micro-CT scanning. ARC reversed IL-1β-induced upregulation of MMP3, MMP13, and COX-2 and the downregulation of collagen II and SOX9. It modulated cholesterol metabolism in IL-1β-stimulated chondrocytes by inhibiting the CH25H-CYP7B1-RORα axis, reducing cartilage damage and proteoglycan loss in OA rats, and effectively inhibiting subchondral bone osteolysis. ARC inhibits IL-1β-induced inflammatory responses and ECM degradation, suggesting its potential as a therapeutic agent for OA. It acts partly by modulating cholesterol metabolism and suppressing the CH25H/CYP7B1/RORα axis in chondrocytes., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: All animal handling procedures were approved by the Animal Welfare Ethics Committee of First Affiliated Hospital of Guangzhou University of Chinese Medicine (K2020-131). We state that all experiments in this study were conducted in accordance with National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Meanwhile, we confirm that our work was carried out in compliance with the ARRIVE guidelines., (© 2024. The Author(s).)

Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The growth plate is the primary site of longitudinal bone growth with chondrocytes playing a pivotal role in endochondral bone development. Chondrocytes undergo a series of differentiation steps, resulting in the formation of a unique hierarchical columnar structure comprising round, proliferating, pre-hypertrophic, and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic stages, are a critical cell population in the growth plate. However, the molecular basis of pre-hypertrophic chondrocytes remains largely undefined. Here, we employed scRNA-seq analysis on fluorescently labeled growth plate chondrocytes for their molecular characterization. Serine incorporator 5 (Serinc5) was identified as a marker gene for pre-hypertrophic chondrocytes. Histological analysis revealed that Serinc5 is specifically expressed in pre-hypertrophic chondrocytes, overlapping with Indian hedgehog (Ihh). Serinc5 represses cell proliferation and Col2a1 and Acan expression by inhibiting the transcriptional activity of Sox9 in primary chondrocytes. Chromatin profiling using ChIP-seq and ATAC-seq revealed an active enhancer of Serinc5 located in intron 1, with its chromatin status progressively activated during chondrocyte differentiation. Collectively, our findings suggest that Serinc5 regulates sequential chondrocyte differentiation from proliferation to hypertrophy by inhibiting Sox9 function in pre-hypertrophic chondrocytes, providing novel insights into the mechanisms underlying chondrocyte differentiation in growth plates., (© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Cerebral ischemia/reperfusion injury (IRI) is pathologically associated with ferroptosis. Dexmedetomidine (Dex) exerts neuroprotective activity after cerebral IRI. Our work focused on probing the pharmacologic effect of Dex on ferroptosis during cerebral IRI and the mechanisms involved. Cerebral IRI models were established by oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). 2,3,5-Triphenyltetrazolium chloride (TTC) staining was utilized to detect cerebral infarct size and mNSS was performed to evaluate neurologic deficits. Brain pathologic changes were analyzed by HE staining. Lipid peroxidation level was detected by C11-BODIPY staining, and Fe 2+ and MDA levels were measured using the kits. Cell vitality was examined by CCK-8 assay. Dual-luciferase reporter and ChIP assays were adopted to determine the interaction between SOX9 and DMT1 promoter. Dex ameliorated ferroptosis and neuronal death induced by MCAO and OGD/R. SOX9 upregulation abolished the inhibitory effect of Dex on OGD/R-induced ferroptosis and neuronal death in SH-SY5Y cells. Our further trials showed that SOX9 transcriptionally activated DMT1 expression. As expected, DMT1 overexpression prevented Dex-induced decrease in ferroptosis and neuronal death in OGD/R-treated SH-SY5Y cells. Dex inhibited ferroptosis to exert neuroprotection effects on cerebral IRI by inactivating the SOX9/DMT1 axis., (© 2025 John Wiley & Sons Ltd.)

Severe damage to the intrahepatic biliary duct (IHBD) network occurs in multiple human advanced cholangiopathies, such as primary sclerosing cholangitis, biliary atresia and end-stage primary biliary cholangitis. Whether and how a severely damaged IHBD network could reconstruct has remained unclear. Here we show that, although the gallbladder is not directly connected to the IHBD, there is a common hepatic duct (CHD) in between, and severe damage to the IHBD network induces migration of gallbladder smooth muscle cells (SMCs) to coat the CHD in mouse and zebrafish models. These gallbladder-derived, CHD-coating SMCs produce retinoic acid to activate Sox9b in the CHD, which drives proliferation and ingrowth of CHD cells into the inner liver to reconstruct the IHBD network. This study reveals a hitherto unappreciated function of the gallbladder in the recovery of injured liver, and characterizes mechanisms involved in how the gallbladder and liver communicate through inter-organ cell migration to drive tissue regeneration. Carrying out cholecystectomy will thus cause previously unexpected impairments to liver health., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Suppressing tumor metastasis is a crucial strategy for improving survival rates in patients with colorectal cancer (CRC), with cancer stem cells (CSCs) being the primary drivers of metastasis. Current therapeutic approaches targeting CSCs are limited, and their molecular mechanisms remain unclear. To address this challenge, a biomimetic nanoparticle delivery system, CMD-BHQ3-PTL/DOX@RBCM is developed, to deliver the stem cell regulator, piceatannol (PTL). This system used carboxymethyl dextran (CMD) and Black Hole Quencher 3 (BHQ3) to encapsulate PTL and the cytotoxic drug doxorubicin (DOX) within a red blood cell membrane (RBCm), enhancing stability and biocompatibility while allowing gradual drug release under hypoxic conditions. The effects of PTL are investigated on CSCs using molecular biology experiments, plasmid construction, and high-throughput sequencing and elucidated the molecular mechanisms underlying this biomimetic nanoparticle delivery system. The therapeutic efficacy of PTL is validated at the tissue level using subcutaneous and metastatic tumor models in human and murine systems. The results demonstrated that CMD-BHQ3-PTL/DOX@RBCM effectively addressed the challenges of specificity and biocompatibility in vivo, significantly inhibiting CSC-related tumor metastasis. This inhibitory effect is closely associated with the Hippo/YAP1/SOX9 pathway. This study highlights the effectiveness of the pH-responsive biomimetic nanoparticle system CMD-BHQ3-PTL/DOX@RBCm in delivering PTL to tumor sites, with SOX9 and its upstream Hippo/YAP1 pathway playing a critical role in the underlying mechanism., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Introduction: Non-small cell lung cancer (NSCLC) is a type of malignant tumor with high morbidity as well as mortality. The process of lung cancer may be driven by cancer stem cells. It was known that MFAP5 enhanced the occurrence of diverse types of cancer. Also, MFAP5 has the potential to induce the degradation of FBW7 which is a tumor suppressor. Lower levels of FBW7 enhance the stability of Sox9, which is the cancer stem cell-related protein. However, whether the MFAP5 can modulate the stem cell features of NSCLC cells by modulating the FBW7/Sox9 axis is unclear. Therefore, this study aimed to explore the role of MFAP5/FBW7/Sox9 axis on the stem cell features of NSCLC cells and develop a new treatment of this carcinoma., Material and Methods: In this study, we explored the effects of MFAP5 on the stem cell features of NSCLC cells for the first time. We established MFAP5 overexpression and knockdown NSCLC cells. Clone formation assays and cell sphere culture assays were conducted for the exploration of the growth and stem cell features of these cells. Western blotting was applied for the detection of Sox9 and FBW7 expression in these cells. CHX was applied for the treatment of these cells for the detection of degradation of Sox9. Finally, we overexpressed the Sox9 in MFAP5 knockdown NSCLC cells., Results: MFAP5 promoted the growth and stem cell features of these cells. Knockdown of MFAP5 induced higher levels of FBW7 while restricting the expression of Sox9. Knockdown of MFAP5 aggravated the degradation of Sox9. Overexpression of Sox9 abrogated the efficacy of MFAP5 inhibition on the growth as well as stem cell features of these cells. The results of this study clarified the role of MFAP5/FBW7/Sox9 axis on the development of non-small cell lung cancer cells, providing the potential therapeutic target for the clinical treatment of NSCLC., Conclusion: MFAP5 maintained the stem cell features of non-small cell lung cancer cells by modulating FBW7/Sox9 axis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Sox17 is a key transcriptional regulator of endoderm formation and function in the gallbladder, blood vessels and reproductive organs. Although multiple transcript variants of Sox17 have been suggested, the precise mechanisms underlying their time- and tissue-specific expression remain unclear. In this study, we discovered two putative regulatory sequences (R1 and R2) adjacent to different transcription start sites of mouse Sox17 exon 1 and generated deletion mice for these regions (Sox17 Δdr/Δdr ). Sox17 Δdr/Δdr mice were alive and fertile, and they possessed a normal-sized gallbladder. However, semiquantitative analysis of immunostaining showed that the expression levels of SOX17 in Sox17 Δdr/Δdr embryos were reduced to less than 50% of the wild-type in the gallbladder epithelium. Furthermore, the bile ductal epithelium marker SOX9 was abnormally upregulated, and PAS/DBA-positive mucin secretion-like epithelial cells were induced in the Sox17 Δdr/Δdr gallbladder. Our results demonstrate that the distal sequence of Sox17, including R1 and R2, is important for the regulation of Sox17 gene expression in the embryonic gallbladder and is crucial for normal gallbladder epithelial development., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Background: Lung cancer is the deadliest form of malignancy and the most common subtype is non-small cell lung cancer (NSCLC). Hypoxia is a typical feature of solid tumor microenvironment. In the current study, we clarified the effects of hypoxia on stemness and metastasis and the molecular mechanism., Methods: The biological functions were assessed using the sphere formation assay, Transwell assay, and XF96 extracellular flux analyzer. The protein levels were detected by western blot. The lactylation modification was assessed by western blot and immunoprecipitation. The role of SOX9 in vivo was explored using a xenografted tumor model., Results: We observed that hypoxia promoted sphere formation, migration, invasion, glucose consumption, lactate production, glycolysis, and global lactylation. Inhibition of glycolysis suppressed cell stemness, migration, invasion, and lactylation. Moreover, hypoxia increased the levels of SOX9 and lactylation of SOX9, whereas inhibition of glycolysis reversed the increase. Additionally, knockdown of SOX9 abrogated the promotion of cell stemness, migration, and invasion. In tumor-bearing mice, overexpression of SOX9 promoted tumor growth, and inhibition of glycolysis suppressed tumor growth., Conclusion: Hypoxia induced the lactylation of SOX9 to promote stemness, migration, and invasion via promoting glycolysis. The findings suggested that targeting hypoxia may be an effective way for NSCLC treatment and reveal a new mechanism of hypoxia in NSCLC.

The intracellular pathogen Chlamydia trachomatis can inflict substantial damage on the host. Notably, Chlamydia infection is acknowledged for its precise modulation of diverse host signaling pathways to ensure cell survival, a phenomenon intricately connected to genetic regulatory changes in host cells. To monitor shifts in gene regulation within Chlamydia-infected cells, we employed mesenchymal stem cells (MSCs) as a naïve, primary cell model. Utilizing biochemical methods and imaging, our study discloses that acute Chlamydia infection in human MSCs leads to the downregulation of transcription factors Oct4, Sox2, and Nanog, suggesting a loss of pluripotency markers. Conversely, pluripotency markers in MSCs were sustained through treatment with conditioned medium from infected MSCs. Additionally, there is an augmentation in alkaline phosphatase activity, along with elevated Sox9 and CD44 mRNA expression levels observed during acute infection. A comprehensive screening for specific cell markers using touchdown PCR indicates an upregulation of mRNA for the early chondrogenesis gene Sox9 and a decrease in mRNA for the MSC marker vimentin. Real-time PCR quantification further corroborates alterations in gene expression, encompassing increased Sox9 and CD44 mRNA levels, alongside heightened alkaline phosphatase activity. In summary, the infection of MSCs with C. trachomatis induces numerous genetic deregulations, implying a potential trend towards differentiation into chondrocytes. These findings collectively underscore a targeted impact of Chlamydia on the gene regulations of host cells, carrying significant implications for the final fate and differentiation of these cells.

The limited self-repair capacity of cartilage due to its avascular and aneural nature leads to minimal regenerative ability. Autologous chondrocyte transplantation (ACT) is a popular treatment for cartilage defects but faces challenges due to chondrocyte dedifferentiation in later passages, which results in undesirable fibroblastic phenotypes. A promising treatment for cartilage injuries and diseases involves tissue engineering using cells (e.g., chondrocytes), scaffolds (e.g., Alginate Sulfate (AlgSulf)), and biochemical signals (e.g., Salidroside and TGF-β). This study focuses on investigating the effects of AlgSulf scaffolds with varying degrees of sulfation, Salidroside, and TGF-β on the proliferation, viability, and phenotype maintenance of chondrocytes. The findings demonstrate that AlgSulf films with a degree of sulfation (DS) = 2, treated with a combination of Salidroside and TGF-β, significantly enhanced chondrocyte proliferation ( p < 0.001 and p < 0.0001 in P2 and P4, respectively), preserved round cell morphology, and maintained cartilage-specific gene expression (Col2, Aggrecans, and SOX9) while downregulating fibroblastic markers (Col1, MMP13, IL-1β, and IL-6). Our findings suggest the potential of this combination for enhancing cartilage regeneration in tissue engineering applications.

Oligodendroglial cells generate myelin sheaths in the vertebrate central nervous system to render rapid saltatory conduction possible and express the highly related Sox8, Sox9 and Sox10 transcription factors. While Sox9 and Sox10 fulfill crucial regulatory roles, Sox8 has only a limited impact on oligodendroglial development and myelination. By replacing Sox10 with Sox8 or Sox9 in the oligodendroglial Oln93 cell line, and comparing the expression profiles, we show here that Sox8 regulates the same processes as Sox10 and Sox9, but exhibits a substantially lower transcriptional activity under standard culture conditions. Sox8 influences fewer genes in their expression and changes their expression level less drastically, despite comparable binding to relevant regulatory regions in oligodendroglial cells. Therefore, it is likely that Sox8 and Sox10 vary in their transcriptional activity because of differences in their interactions with partner proteins. Intriguingly, it is the aminoterminal one third of the Sox protein that is responsible for the differential activities of Sox8 and Sox10, rather than the carboxyterminal two thirds that contain the known transactivation domains. Our study aims to provide an understanding of the relationship of Sox8 and Sox10 as paralogous transcription factors and their degree of functional redundancy in oligodendroglial cells, along with implications for health and disease.

Production of melanin pigments is a protective mechanism of the skin against ultraviolet (UV)-induced damage and carcinogenesis. However, the molecular basis for melanogenesis is still poorly understood. Herein, we demonstrate a critical interplay between the primary cilium and the melanocortin 1 receptor (MC1R) signaling. Our data show that UV and α-melanocyte-stimulating hormone (α-MSH) trigger cilium formation in human melanocytes and melanoma cells. Deficiency of MC1R or the presence of its red hair color (RHC) variations significantly attenuates the UV/α-MSH-induced ciliogenesis. Further investigation reveals that MC1R enters the cilium upon UV/α-MSH stimulation, which is facilitated by the interaction of MC1R with the BBSome and the palmitoylation of MC1R. MC1R interacts with the BBSome through the second and third intercellular loops, which contain the common RHC variant alleles (R151C and R160W). These RHC variants of MC1R exhibit attenuated ciliary localization, and enforced ciliary localization of these variants elevates melanogenesis. Ciliary MC1R triggers a sustained cAMP signaling and selectively stimulates Sox9, which appears to up-regulate melanogenesis-related genes as the transcriptional cofactor for MITF. These findings reveal a previously unrecognized nexus between MC1R and cilia and suggest an important mechanism for RHC variant-related pigmentary defects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Diabetes mellitus can induce liver injury and easily progress to liver fibrosis. However, there is still a lack of effective treatments for diabetes-induced hepatic fibrosis. Cordycepin (COR), a natural nucleoside derived from Cordyceps militaris, has demonstrated remarkable efficacy in treating metabolic diseases and providing hepatoprotective effects. However, its protective effect and underlying mechanism in diabetes-induced liver injury remain unclear. This study utilized a high-fat diet/streptozotocin-induced diabetic mouse model, as well as LX-2 and AML-12 cell models exposed to high glucose and TGF-β1, to explore the protective effects and mechanisms of Cordycepin in liver fibrosis associated with diabetes. The results showed that COR lowered blood glucose levels, enhanced liver function, mitigated fibrosis, and suppressed HSC activation in diabetic mice. Mechanistically, COR attenuated the activation of the Wnt/β-catenin pathway by inhibiting β-catenin nuclear translocation, and β-catenin knockdown further intensified this effect. Meanwhile, COR significantly inhibited SOX9 expression in vivo and in vitro. Knockdown of SOX9 downregulated Wnt3a and β-catenin expression at the protein and gene levels to exacerbate the inhibitory action of COR on HG&TGF-β1-induced HSCs activations. These results indicate SOX9 is involved in the mechanism by which COR deactivates the Wnt/β-catenin pathway in hepatic fibrosis induced by diabetes. Moreover, prolonged half-life time, slower metabolism and higher exposure of COR were observed in diabetes-induced liver injury animal model via pharmacokinetics studies. Altogether, COR holds potential as a therapeutic agent for ameliorating hepatic injury and fibrosis in diabetes by suppressing the activation of the SOX9-mediated Wnt/β-catenin pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gallbladder cancer (GBC) is a highly aggressive malignancy lacking clinically available targeted therapeutic agents. Super-enhancers (SEs) are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in cancers. To study SE in GBC, the genomic distribution of H3K27ac is profiled in multiple GBC tissue and cell line samples to establish the SE landscape and its associated core regulatory circuitry (CRC). The biliary lineage factor SOX9 and Wnt pathway effector TCF7L2, two master transcription factor (TF) candidates identified by CRC analysis, are verified to co-occupy each other's SE region, forming a mutually autoregulatory loop to drive oncogenic SE reprogramming in a subset of GBC. The SOX9/TCF7L2 double-high GBC cells are highly dependent on the two TFs and enriched of SE-associated gene signatures related to stemness, ErbB and Wnt pathways. Patients with more such GBC cells exhibited significantly worse prognosis. Furthermore, SOX9/TCF7L2 double-high GBC preclinical models are found to be susceptible to SE-targeted CDK7 inhibition therapy in vitro and in vivo. Together, this study provides novel insights into the epigenetic mechanisms underlying the oncogenesis of a subset of GBCs with poorer prognosis and illustrates promising prognostic stratification and therapeutic strategies for treating those GBC patients in future clinical trials., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Objective: Loose bodies are free-floating tissues of cartilage and bone that can cause pain, swelling, the inability to straighten the knee, or intermittent locking of the knee. Loose bodies can arise from degenerative joint disease, flake fractures, osteochondritis dissecans, or chondromatosis. We hypothesized that loose bodies can be classified in stages with tissue characteristics similar to endochondral ossification., Design: Loose bodies were harvested from patients undergoing joint replacement. Samples were processed for histology, gene expression analysis, and micro-computed tomography (µCT). Cartilage- and bone-related genes and proteins were selected for immunofluorescence stainings (collagen type I, II, and X, SOX9 [SRY-box transcription factor 9], and MMP13 [matrix metalloproteinase 13]) and gene expression analysis ( FN [fibronectin], COL1A1, COL2A1, COL10A1, SOX9, MMP13 , and aggrecan [ ACAN ])., Results: Loose bodies were grouped in 4 stages: fibrous, (mineralized) cartilaginous, cartilage and bone, and bone. Hyaline-like cartilage tissue with Benninghoff arcades was present in stages 2 and 3. A transition from cartilaginous to mineralized tissue and bone trabecula was defined by an increase in COL1A1 and COL10A1 (stage 3 vs. 4: p = 0.047) positive area. Stage 4 showed typical trabecular bone tissue. The relative volume of calcified tissue (mineralized cartilage and bone tissue) decreased with stages (stages 1-2 vs. 3: p = 0.002; stage 1-2 vs. 4: p = 0.012). COL2A1 expression and stained area decreased from stages 1-2 to 4 ( p = 0.010 and p = 0.004). ACAN expression decreased from stage 1-2 to stage 3 ( p = 0.049) and stage 4 ( p = 0.002)., Conclusion: Loose bodies show tissue characteristics similar to endochondral ossification. They are probably a relevant substrate for regenerative therapeutic interventions in joint disease., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Furan, the basic skeleton of various flavoring agents, induces cholangiocellular tumors with higher incidences in the caudate lobe and hepatocellular tumors without the lobe specificity in rats, but the mechanism is unclear. We investigated the lobe distribution of possible carcinogenic events. Furan caused proliferation/infiltration of oval and inflammatory cells prominently in the caudate lobe as early as 4 weeks and cholangiofibrosis in this lobe at 8 weeks. In vivo mutagenicity assays using DNA extracted from the caudate or left lateral lobe of male gpt delta rats, the reporter gene-transgenic rats, treated with 8 mg/kg furan for 4 or 8 weeks showed negative outcomes. The distribution of glutathione S-transferase placental form (GST-P)-positive or sex-determining region Y-box 9 (SOX9)-positive hepatocytes was examined. Significant increases in the number of GST-P-positive hepatocytes were observed in all lobes of furan-treated rats at 8 weeks. By contrast, SOX9-positive hepatocytes, liver injury-inducible progenitor cells, were also found in all lobes of treated rats, the incidences of which were by far the highest in the caudate lobe. In addition, some of these hepatocytes also co-expressed delta like 1 homolog (DLK1), a hepatoblast marker, particularly in areas with a predominant presence of inflammatory cells. Overall, furan induced liver injury, leading to the appearance of SOX9-positive hepatocytes, some of which were subjected to dedifferentiation in the inflammatory microenvironment of a cholangiocarcinoma-prone lobe. Thus, the appearance of SOX9-positive hepatocytes together with GST-P-positive hepatocytes could be initial events in furan-induced hepatocarcinogenesis via non-genotoxic mechanisms., (© 2024 John Wiley & Sons Ltd.)

Specific spatiotemporal patterns of the normal glial differentiation during human brain development have not been thoroughly studied. Immunomorphological studies on postmortem material have remained a basic method for human neurodevelopmental studies so far. The main problem for the immunohistochemical research of astrogliogenesis is that now there are no universal astrocyte markers, that characterize the whole mature astrocyte population or precursors at each stage of development. To define the general course of astrogliogenesis in the developing human cortex, 25 fetal autopsy samples at the stages from eight postconceptional weeks to birth were collected for the immunomorphological analysis. Spatiotemporal immunoreactivity patterns with the panel of markers (ALDH1L1, GFAP, S100, SOX9, and Olig-2), related to glial differentiation were described and compared. The early S100 + cell population of ventral origin was described as well. This S100 + cell distribution deviated from the SOX9-immunoreactivity pattern and was similar to the Olig-2 one. In the given material the dorsal gliogenic wave was characterized by ALDH1L1-, GFAP-, and S100-immunoreactivity manifestation in the dorsal proliferative niche at the end of the early fetal period. The time point of dorsal astrogliogenesis was agreed upon not later than the 17 GW stage. ALDH1L1 + , GFAP + , S100 + , and SOX9 + cell expansion patterns from the ventricular and subventricular zones to the intermediate zone, subplate, and cortical plate were described at the end of early fetal, middle, and late fetal periods. The ALDH1L1-, GFAP-, and S100-immunoreactivity patterns were shown to be not completely identical., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Native cartilage has low capacity for regeneration because it has very few progenitor cells. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and human umbilical cord-derived MSCs (hUC-MSCs) have been employed as promising sources of stem cells for cartilage injury repair. Reproduction of hyaline cartilage from MSCs remains a challenging endeavor. The paracrine factors secreted by chondrocytes possess the capability to induce chondrogenesis from MSCs., Methods: The conditioned medium derived from chondrocytes was utilized to induce chondrogenic differentiation of hUCB-MSCs and hUC-MSCs. The expression levels of collagen type I alpha 1 chain (Col1a1), collagen type II alpha 1 chain (Col2a1), and SRY-box transcription factor 9 (SOX9) were assessed through quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays. To elucidate the mechanism of differentiation, the concentration of transforming growth factor-β1 (TGF-β1) in the conditioned medium of chondrocytes was quantified using enzyme-linked immunosorbent assay (ELISA). Meanwhile, the viability of cells was assessed using Cell Counting Kit-8 (CCK-8) assays., Results: The expression levels of Col2a1 and SOX9 were found to be higher in induced hUC-MSCs compared to those in induced hUCB-MSCs. The conditioned medium of chondrocytes contained TGF-β1. The CCK-8 assays revealed that the proliferation rate of hUC-MSCs was significantly higher compared to that of hUCB-MSCs., Conclusions: The chondrogenic potential and proliferation capacity of hUC-MSCs surpass those of hUCB-MSCs, thereby establishing hUC-MSCs as a superior source of seed cells for cartilage tissue engineering., (© 2024 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Current treatment approaches cannot exactly regenerate cartilage tissue. Regarding some problems encountered with cell therapy, exosomes are advantageous because of their "cell-free" nature. This study examines the relationship between IL-10 and TGF-β and Canonical Wnt/β-catenin signal pathways in human adipose tissue-derived MSCs exosomes (hAT-MSCs-Exos) after in vitro chondrogenic differentiation. Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and, as a control group, human fetal chondroblast cells (hfCCs) were differentiated chondrogenically in vitro. Exosome isolation and characterization analyses were performed. Chondrogenic differentiation was shown by Alcian Blue and Safranin O stainings. The expression levels of IL-10, TGF-β/SMAD signaling pathway genes, and Canonical Wnt/β-catenin signaling pathway genes, which play an essential role in chondrogenesis, were analyzed by RT-qPCR. Conditioned media cytokine levels were measured by using the TGF-β and IL-10 ELISA kits. IL-10 expression was upregulated in both chondrogenic differentiated hAT-MSC-Exos (dhAT-MSC-Exos) (p < 0.0001). In the TGF-β signaling pathway, TGF-β (p < 0.0001), SMAD2 (p < 0.0001), SMAD4 (p < 0.001), ACAN (p < 0.0001), SOX9 (p < 0.05) and COL1A2 (p < 0.0001) expressions were upregulated in dhAT-MSC-Exos. SMAD3 expression was upregulated in non-differentiated hAT-MSC-Exos. In the Canonical Wnt/β-catenin signaling pathway, WNT (p < 0.0001) and CTNNB1(p < 0.0001) expressions were upregulated in dhAT-MSC-Exos. AXIN (p < 0.0001) expression was upregulated in non-differentiated hAT-MSC-Exos. TGF-β and IL-10 levels were higher in dhAT-MSCs) (p < 0.0001). Related to these results, IL-10 may induce TGF-β/SMAD and Canonical Wnt/β-catenin signaling pathways in hAT-MSC exosomes obtained after chondrogenic differentiation. Therefore, using these exosomes for cartilage regeneration can lead to the development of treatment methods., Competing Interests: Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest. Consent for Publication All the authors approved the publication., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background/aim: Stem-like cancer cells are believed to be the leading cause of therapy resistance in malignant melanoma (MM). All-trans retinoic acid (ATRA) differentiation therapy is considered a promising approach to eradicate stem-like cancer cells, but some melanoma cells are resistant to ATRA. This study aimed to examine whether resveratrol (RS), a natural polyphenol compound, could improve the response of MM stem-like cells to ATRA and explore the possible underlying mechanisms., Materials and Methods: MM stem-like cells were established from a spheroid model of A375 human MM cell line. The response to RES alone and in combination with ATRA, was examined through analysis of cancer stemness, cell viability, and protein expression., Results: The stem-like cells showed resistance to the anticancer drug docetaxel; however, the combination of RES and ATRA augmented the effects of docetaxel. Accordingly, these combinatorial effects were associated with significant inhibition of the expression levels of stemness markers CD133, OCT4, CD271, and ABCG2. The tested combinations also led to a significant increase in melanocyte differentiation marker SOX9, while efficiently suppressing the dedifferentiation marker SOX10. Notably, RES alone effectively up-regulated retinoic acid receptor beta (RARβ) expression and down-regulated crucial mediators like DNMT1, polycomb-group proteins EZH2, and BMI-1, which mechanistically explain how RES enhanced the differentiation-inducing effects of ATRA., Conclusion: The resistance of MM stem-like cells to ATRA can be attenuated by RES and combined applications of ATRA and RES provide a promising strategy for MM treatment., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The transcription factor SOX9 is integral to tissue homeostasis and is implicated in skeletal malformation, campomelic dysplasia, and osteoarthritis (OA). Despite extensive research, the complete regulatory landscape of SOX9 transcriptional activity, interconnected with signaling pathways (TGFβ, WNT, BMP, IHH, NFκB, and HIF), remains challenging to decipher. This study focuses on elucidating SOX9 signaling in OA pathology using Fluorescence Recovery After Photobleaching (FRAP) to assess SOX9 activity directly in live human primary chondrocytes (hPCs). Single cell FRAP data revealed two distinct subpopulations with differential SOX9 dynamics, showing varied distribution between healthy and OA hPCs. Moreover, inherently elevated SOX9-DNA binding was observed in healthy hPCs compared to preserved and OA counterparts. Anabolic factors (BMP7 and GREM1) and catabolic inhibitors (DKK1 and FRZb) were found to modulate SOX9 transcriptional activity in OA-hPCs. These findings provide valuable insights into the intricate regulation of SOX9 signaling in OA, suggesting potential therapeutic avenues for modulating SOX9 activity in diseased states., (© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.)

In mammals, male sexual development is initiated by the expression of the Sex-determining-Region-Y (SRY) gene. SRY contains a highly conserved high mobility group (HMG) box essential for DNA binding and activity. Variants in SRY cause Differences of Sex Development (DSD), accounting for 10-15% of 46, XY gonadal dysgenesis cases. Here, we present the functional analysis of five SRY coding variants identified in patients with 46, XY DSD. Four variants (p.Asp58Glu, p.Arg75Lys, p.Met85Thr, and p.Arg86Ter) are located within the HMG box and one variant (p.Tyr198Cysfs∗18) located in the C-terminal domain. We functionally characterise the impact of these variants in vitro, investigating SRY localisation and transactivational activity using SOX9 regulatory elements that are responsive to SRY. We find that three variants (p.Met85Thr, p.Arg86Ter, and p.Tyr198Cysfs∗18) have reduced or abolished transactivational activity suggesting these are pathogenic, with the p.Arg86Ter variant undetectable in our assays and the p.Met85Thr variant exhibiting reduced nuclear localisation. The pathogenic mechanisms underlying reduced activity of the novel elongated p.Tyr198Cysfs∗18 variant is however unclear, although this variant also affected localisation. In contrast, two additional variants (p.Asp58Glu and p.Arg75Lys) had no discernible effects on nuclear localisation or transactivational activity despite in silico analysis predicting impaired DNA binding. Taken together, our data establish the likely pathogenicity of these SRY variants and improve diagnostic certainty for the patients in which they were identified., Competing Interests: Declaration of competing interest The authors have nothing to declare., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Normal ovarian function requires the expression of estrogen receptors α (ESR1) and β (ESR2) in distinct cell types within the ovary. The double estrogen receptor knockout (αβERKO) ovary had the appearance of seminiferous tubule-like structures that expressed SOX9; this phenotype was lost when the animals were repeatedly backcrossed to the C57BL/6J genetic background. A new line of ERKO mice, Ex3αβERKO, was developed for targeted disruption on a mixed genetic background. Histological examination of the ovaries in the Ex3αβERKO showed the appearance of seminiferous tubule-like structures in mice aged 6 to 12 months. These dismorphogenic regions have cells that no longer express granulosa cell-specific FOXL2, while other cells express Sertoli cell-specific SOX9 as examined by immunohistochemistry. Whole ovarian gene expression analysis in Ex3αERKO, Ex3βRKO, and Ex3αβERKO found many genes differentially expressed compared to controls with one Esr1 and Esr2 allele. The genes specific to the Ex3αβERKO ovary were compared to other models of postnatal ovarian transdifferentiation, identifying 21 candidate genes. To examine the genetic background contributions, DNA was isolated from αβERKO mice that did not show ovarian transdifferentiation and compared to DNA from Ex3αβERKO using Mouse Diversity Array. A genomic region putatively associated with transdifferentiation was identified on Chr18 (5-15 M) and genes in this region were compared to the genes differentially expressed in models of ovarian transdifferentiation. This work demonstrates the importance of ESRs in maintaining granulosa cell differentiation within the ovary, identifies several potential gene candidates, and suggests that genetic background can be a confounding factor., (Published by Oxford University Press on behalf of the Endocrine Society 2024.)

Although hyperplasia of the anorectal transitional zone (TZ) has been reported in mouse models of ulcerative colitis, the mechanisms underlying this phenomenon are not fully understood. We characterized keratin subtypes and examined the expression of stem cell markers in the TZ. Dextran sodium sulfate-treated mice showed abnormal repair of the anorectal region, which consisted of mixed hyperplastic TZ and regenerating crypts. Liquid chromatography-tandem mass spectrometry from the paraffin-embedded TZ in the treated mice revealed that the major keratins were type I cytokeratin (CK)13 and type II CK5, but notable expression of type I CK10 and CK42 and type II CK1, CK4, CK6a, CK8, and CK15 was also detected. Hyperplastic TZ was characterized by the expression of tumor protein 63, sex-determining region Y-box 2 (SOX2), SOX9, and leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5). Lgr5 was highly expressed in the TZ in the early stages of colitis, followed by higher expression levels of SOX2. The TZ-derived organoids expressed LGR5, SOX2, and SOX9. The present study suggests that transitional zones showing abnormal keratin assembly and stem cell activation may interfere with rectal crypt regeneration, leading to pathological anorectal remodeling in severe colitis.

Zinc is the second most abundant trace element in the human body, stored mainly in the bones. Zinc is required for bone growth and homeostasis and is also a crucial cofactor for numerous proteins that play key roles in maintaining microstructural integrity and bone remodeling. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent progenitors found in the bone marrow stroma and can differentiate along multiple lineage pathways. In this study, we investigated the effect of zinc on the osteogenic differentiation of BMSCs. We stimulated the osteogenic differentiation of BMSCs with high phosphate and Ca-containing osteogenic medium (PiCa) in the presence or absence of zinc. We followed calcification by measuring ECM mineralization, the Ca content of the ECM, mRNA, and the protein expression of the osteo-chondrogenic transcription factor RUNX2 and SOX9 and its targets OCN and ALP. Zinc dose-dependently abolished PiCa-induced ECM mineralization and decreased the expression of RUNX2, SOX9, OCN, and ALP. Serum albumin did not alter the inhibitory effect of zinc on BMSC mineralization. Our further analysis with the zinc-chelator TPEN and ZnCl 2 confirmed the specific inhibitory effect of free zinc ions on BMSC mineralization. Zinc inhibited phosphate uptake and PiCa-induced upregulation of the sodium-dependent phosphate cotransporters (PiT-1 and PiT-2). Zinc attenuated the PiCa-induced increase in ROS production. Taken together, these data suggest that zinc inhibits PiCa-induced BMSC calcification by regulating phosphate uptake and ROS production.

Fibrosis results from the excessive production of extracellular matrix proteins by myofibroblasts. It has recently been reported that in the heart, myofibroblasts develop chondrocyte-like properties following myocardial infarction as fibrosis progresses and tissues stiffen. However, the nature of these chondrocyte-like myofibroblasts remains unclear. In this study, we found that the expression of the proline- and arginine-rich end leucine-rich repeat protein (PRELP) was upregulated in hearts and livers stiffened by fibrosis with chronic inflammation. Moreover, we established that Prelp was specifically expressed in chondrocyte-like myofibroblasts. Prelp expression was found to be regulated by the transcription factor SOX9, and in cardiac and liver myofibroblasts, Prelp-knockdown was observed to reduce collagen expression. These findings reveal that PRELP is specifically expressed in chondrocyte-like myofibroblasts and that it promotes collagen production. PRELP could thus serve as a novel therapeutic target for treating fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Mandibular retraction is a prevalent dental and maxillofacial deformity that negatively affects patients' functional health and facial aesthetics. It has been challenging to achieve optimal outcomes for patients who have passed the peak of growth and development using only functional orthopedic treatment. There is a pressing need to explore innovative methods to promote the adaptive remodeling of adult condylar cartilage and the mandible in response to external stimuli. This study aimed to investigate the impact of varying injection frequencies of stem cells from the apical papilla (SCAPs) on the growth and development of condylar cartilage and the mandible, as well as their potential for adaptive remodeling., Methods: The study was conducted on 8-week-old adult male Sprague-Dawley rats. The effects of SCAPs injection and different durations of mandibular advancement (MA) on the adaptive remodeling of condylar cartilage and the mandible were assessed. After the initial experimental findings, various injection frequencies of SCAPs were applied to determine the most effective conditions for promoting the growth and adaptive remodeling of condylar cartilage and the mandible during an 8-week period of mandibular advancement., Results: The study found that rats with extended mandibular lead times (8 weeks) or an appropriately increased frequency of mandibular leading time (once every 2 weeks or once every 1 week) exhibited increased lengths of the mandibular body and ascending branch, and a thickened full layer of condylar cartilage. The highest proportions of the proliferative layer, mature layer, and hypertrophic layer were observed in these rats. Additionally, there was a significant increase in the expression levels of SOX9 and COL2A1., Conclusion: The data from this study suggest that adult rats, even after missing their peak growth period, retain the potential for continued growth and development of their condylar cartilage. By prolonging the duration of mandibular advancement and administering injections of stem cells from the apical papilla (SCAPs), it is possible to stimulate the growth and development of the mandibular condyle., Competing Interests: Declarations Ethics approval and consent to participate All human cell cultures and animal experiments conducted in this study were approved by the Ethics Committees of the Jinan University (No. JNUKY-2023–0110). The approved project was titled “Construction and orthodontic function evaluation of 3D Printed Autologous Tooth-Bone transformation prosthesis based on CEMP1 regulating periodontal regeneration mechanism.” The date of approvals is December 29, 2023. Written informed consents were obtained from patients and their families who provided teeth for further research. Consent for publication Not applicable. Competing interests The authors have declared no competing interests., (© 2024. The Author(s).)

Long noncoding RNAs (lncRNAs) are involved in the regulation of triple-negative breast cancer (TNBC) senescence, while pro-carcinogenic lncRNAs resist senescence onset leading to the failure of therapy-induced senescence (TIS) strategy, urgently identifying the key senescence-related lncRNAs (SRlncRNAs). We mined seven SRlncRNAs (SOX9-AS1, LINC01152, AC005152.3, RP11-161 M6.2, RP5-968 J1.1, RP11-351 J23.1 and RP11-666A20.3) by bioinformatics, of which SOX9-AS1 was reported to be pro-carcinogenic. In vitro experiments revealed the highest expression of SOX9-AS1 in MDA-MD-231 cells. SOX9-AS1 knockdown inhibited cell growth (proliferation, cycle and apoptosis) and malignant phenotypes (migration and invasion), while SOX9-AS1 overexpression rescued these effects. Additionally, SOX9-AS1 knockdown facilitated tamoxifen-induced cellular senescence and the transcription of senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-1β, IL-6 and IL-8) mechanistically by resisting senescence-induced Wnt signal (GSK-3β/β-catenin) activation. Immune infiltration analysis revealed that low SOX9-AS1 expression was accompanied by a high infiltration of naïve B cells, CD8 + T cells and γδ T cells. In conclusion, SOX9-AS1 resists TNBC senescence via regulating the Wnt signalling pathway and inhibits immune infiltration. Targeted inhibition of SOX9-AS1 enhances SASP and thus mobilises immune infiltration to adjunct TIS strategy., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

In mammals, sex determination is governed by the SRY gene on the Y chromosome, redirecting gonadal development from forming ovaries to testes. Mutations or alterations in the SRY gene can significantly affect phenotypic changes and lineage-specific markers. This study aims to elucidate the role of the SRY gene in buffalo embryos using CRISPR-Cas9 technology. We designed a crRNA targeting the HMG domain of the SRY gene using the CRISPOR algorithm. Nucleofection of sgRNA-Cas9 RNPs into buffalo fibroblasts confirmed efficient cleavage at the targeted site. Using this validated guide, we investigated the role of the SRY gene in sexual determination by electroporating CRISPR-Cas9-RNPs into single-stage zygotes of buffalo. Genetic changes in the SRY gene were confirmed through sequencing, revealing mosaic blastocysts with multiple alleles and non-mosaic mutants. Mutations in SRY gene increased the expression of female lineage-specific gene Wnt4 whereas decreased the expression of male specific gene SOX9 in blastocysts, suggesting reprogramming towards female sex determination pathways. Our findings provide insights into buffalo sex differentiation mechanisms and potential applications in reproductive strategies for breeding programmes., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Uncovering mechanisms of endogenous regeneration and repair through resident stem cell activation will allow us to develop specific therapies for injuries and diseases by targeting resident stem cell lineages. Sox9 + stem cells have been reported to play an essential role in acute kidney injury (AKI). However, a complete view of the Sox9 + lineage was not well investigated to accurately elucidate the functional end state and the choice of cell fate during tissue repair after AKI. To identify the mechanisms of fate determination of Sox9 + stem cells, we set up an AKI model with prostaglandin E2 (PGE 2 ) treatment in a Sox9 lineage tracing mouse model. Single-cell RNA sequencing (scRNA-seq) was performed to analyse the transcriptomic profile of the Sox9 + lineage. Our results revealed that PGE 2 could activate renal Sox9 + cells and promote the differentiation of Sox9 + cells into renal proximal tubular epithelial cells and inhibit the development of fibrosis. Furthermore, single-cell transcriptome analysis demonstrated that PGE 2 could regulate the restoration of lipid metabolism homeostasis in proximal tubular epithelial cells by participating in communication with different cell types. Our results highlight the prospects for the activation of endogenous renal Sox9 + stem cells with PGE 2 for the regenerative therapy of AKI., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Objective: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process., Design: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway., Results: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9., Conclusions: This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cartilage rarely heals spontaneously once damaged. Osteoarthritis (OA) is the most common degenerative joint disease among the elderly; however, effective treatment for OA is currently lacking. Autologous chondrocyte implantation (ACI), an innovative regenerative technology involving the implantation of healthy chondrocytes, may restore damaged lesions. Chondrocytes for ACI may potentially be induced from differentiated somatic cells using retrovirus (RV)-mediated transduction of three reprogramming factors (SOX9, KLF4, and c-MYC). However, the efficiency of the current induction system needs to be improved and the safety issues arising from the genomic integration of the vector DNA have to be addressed. To solve these problems, we used an RNA vector, termed the replication-defective and persistent Sendai virus vector (SeVdp), to express reprogramming factors for chondrocyte induction. Our results showed that the SeVdp-based vector induced chondrocytes more efficiently than the RV vector, probably because of robust and rapid expression of the transgenes, without any apparent integration of the SeVdp vector. The induced chondrocytes formed cartilage-like tissues when injected subcutaneously into mice. Thus, the SeVdp-based system for inducing chondrocytes may act as a foundation for developing safer and more effective treatments for damaged cartilage., (© 2024. The Author(s).)

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. This study investigates the therapeutic potential of secretome derived from adipose tissue mesenchymal stem cells (ASCs) in mitigating inflammation and promoting cartilage repair in an in vitro model of OA. Our in vitro model comprised chondrocytes inflamed with TNF. To assess the therapeutic potential of secretome, inflamed chondrocytes were treated with it and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and extracellular matrix markers were measured. In addition, secretome-treated chondrocytes were subject to a microarray analysis to determine which genes were upregulated and which were downregulated. Treating TNF-inflamed chondrocytes with secretome in vitro inhibits the NF - κB pathway, thereby mediating anti-inflammatory and anti-catabolic effects. Additional protective effects of secretome on cartilage are revealed in the inhibition of hypertrophy markers such as RUNX2 and COL10A1, increased production of COL2A1 and ACAN and upregulation of SOX9 . These findings suggest that ASC-derived secretome can effectively reduce inflammation, promote cartilage repair, and maintain chondrocyte phenotype. This study highlights the potential of ASC-derived secretome as a novel, non-cell-based therapeutic approach for OA, offering a promising alternative to current treatments by targeting inflammation and cartilage repair mechanisms.

Neurofibromatosis type 1 (NF1) is caused by NF1 gene mutations. Patients with NF1 often have complications with tumors, such as neurofibroma. In order to investigate the pathogenesis of human neurofibroma, a systematic comparison of protein expression levels between Schwann cell-like sNF96.2 cells, which originated from malignant peripheral nerve sheath tumors (MPNST), and normal Schwann cells was performed using 4-D label-free proteomic analysis. In addition, the expression levels and localization of dysregulated proteins were confirmed using a Gene Expression Omnibus (GEO) transcriptomic dataset, Western blot analysis, and immunofluorescence labeling. The effects of SRY-box transcription factor 9 (SOX9) in the neurofibroma and surrounding microenvironment were evaluated in vivo using a tumor transplantation model. The present study observed that SOX9 and procollagen C-endopeptidase enhancer (PCOLCE) were significantly altered. NF1 mutation promoted the nuclear translocation and transcriptional activity of SOX9 in neurofibromas. SOX9 increased collagen VI secretions by enhancing the activation of PCOLCE in neurofibroma cells. These findings might provide new perspectives on the pathophysiological significance of SOX9 in neurofibromas and elucidate a novel molecular mechanism underlying neurofibromas., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Although enhanced fibroblast growth factor (FGF) signaling has been demonstrated to be crucial in many cases of syndromic cleft palate caused by tongue malposition in humans, animal models that recapitulate this phenotype are limited, and the precise mechanisms remain elusive. Mutations in FGF9 with the effect of either loss- or gain-of-function effects have been identified to be associated with cleft palate in humans. Here, we generated a mouse model with a transgenic Fgf9 allele specifically activated in cranial neural crest cells, aiming to elucidate the gain-of-function effects of Fgf9 in palatogenesis. We observed cleft palate with 100% penetrance in mutant mice. Further analysis demonstrated that no inherent defects in the morphogenic competence of palatal shelves could be found, but a passively lifted tongue prevented the elevation of palatal shelves, leading to the cleft palate. This tongue malposition was induced by posterior spatial confinement that was exerted by temporomandibular joint (TMJ) dysplasia characterized by a reduction in Sox9+ progenitors within the condyle and a structural decrease in the posterior dimension of the lower jaw. Our findings highlight the critical role of excessive FGF signaling in disrupting spatial coordination during palate development and suggest a potential association between palatal shelf elevation and early TMJ development.

Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by pulmonary vascular remodeling (PVR), primarily due to the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This study aimed to investigate the role and molecular mechanism of SOX9 in hypoxic PH in rats. The findings revealed that SOX9 was upregulated in the pulmonary arteries and PASMCs of hypoxia-exposed rats. SOX9 knockdown inhibited hypoxia-induced proliferation and migration of PASMCs, reduced PVR, and subsequently alleviated hypoxia-induced PH in rats, suggesting that SOX9 plays a critical role in PH. Further investigation demonstrated that SOX9 interacted with DPP4, preventing its ubiquitin degradation in hypoxia-exposed PASMCs. DPP4 knockdown inhibited hypoxia-induced PASMC proliferation and migration, and administration of the DPP4 inhibitor sitagliptin (5 mg/kg) significantly reduced PVR and alleviated hypoxia-induced PH in rats, indicating that SOX9 contributes to PH by stabilizing DPP4. The results also showed that hypoxia induced YAP1 expression and dephosphorylation, leading to YAP1 nuclear localization. YAP1 knockdown promoted the degradation of HIF-1α in hypoxia-exposed PASMCs and inhibited hypoxia-induced proliferation and migration of PASMCs. Additionally, HIF-1α, as a transcription factor, promoted SOX9 expression by binding to the SOX9 promoter in hypoxia-exposed PASMCs. In conclusion, hypoxia promotes the proliferation and migration of PASMCs through the regulation of the YAP1/HIF-1α/SOX9/DPP4 signaling pathway, leading to PH in rats. These findings suggest that SOX9 may serve as a potential prognostic marker and therapeutic target for PH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study investigated the efficacy of using chitosan/alginate nanoparticles loaded with recombinant human bone morphogenetic-2 (rhBMP-2) and SMAD4 encoding plasmid to enhance the chondrogenesis of human bone marrow mesenchymal stem cells (hBM-MSCs) seeded on an extracellular matrix (ECM). The research treatments included the stem cells treated with the biological cocktail (BC), negative control (NC), hBM-MSCs with chondrogenic medium (MCM), hBM-MSCs with naked rhBMP-2 and chondrogenic medium (NB/C), and hBM-MSCs with naked rhBMP-2 and chondrogenic medium plus SMAD4 encoding plasmid transfected with polyethyleneimine (PEI) (NB/C/S/P). The cartilage differentiation was performed with real-time quantitative PCR analysis and alizarin blue staining. The data indicated that the biological cocktail (BC) exhibited significantly higher expression of cartilage-related genes compared to significant differences with MCM and negative control (NC) on chondrogenesis. In the (NB/C/S/P), the expression levels of SOX9 and COLX were lower than those in the BC group. The expression pattern of the ACAN gene was similar to COL2A1 changes suggesting that it holds promising potential for cartilage regeneration., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)