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Myelin is an electrical insulator that enables saltatory nerve conduction and is essential for proper functioning of the central nervous system (CNS). It is formed by oligodendrocytes (OLs) in the CNS, and during OL development various molecules, including extracellular matrix (ECM) proteins, regulate OL differentiation and myelination; however, the role of ECM proteins in these processes is not well understood. Our present work is centered on the analyses of the expression and function of fibulin-7 (Fbln7), an ECM protein of the fibulin family, in OL differentiation. In the expression analysis of Fbln7 in the CNS, we found that it was expressed at early postnatal stage and localized in the processes of OL precursor cells (OPCs), in the inner region of myelin, and in axons. The functional analysis using recombinant Fbln7 protein (rFbln7) revealed that rFbln7 promoted OPC attachment activity via β1 integrin and heparan sulfate receptors. Further, rFbln7 induced the adhesion to neurites and the differentiation of OLs. Altogether, our results show that Fbln7 promotes the adhesion between OLs and axons and OL differentiation., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Myoblasts play a critical role in the regeneration of skeletal muscle following injury. It has been reported that local elevation of transforming growth factor-β1 (TGF-β1) after skeletal muscle injury induces differentiation of myoblasts into myofibroblasts. However, the mechanisms underlying this differentiation process remain incompletely understood. In this study, we found that Fibulin2 expression significantly increases in myoblasts in response to TGF-β1 stimulation. Elevated Fibulin2 levels enhance the expression of fibrotic markers. Conversely, downregulation of Fibulin2 in myoblasts inhibits the upregulation of fibrotic markers induced by TGF-β1 stimulation. Extracellular secretion of Fibulin2 activates the TGF-β1-Smad2 pathway, thereby promoting the upregulation of fibrotic markers. Hence, Fibulin2 and TGF-β1 form a positive feedback loop that facilitates differentiation of myoblasts into myofibroblasts., 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. Published by Elsevier B.V.)

The mammalian Hippo kinases, MST1 and MST2, regulate organ development and suppress tumor formation by balancing cell proliferation and death. In macrophages, inflammasomes detect molecular patterns from invading pathogens or damaged host cells and trigger programmed cell death. In addition to lytic pyroptosis, the signatures associated with apoptosis are induced by inflammasome activation, but how the inflammasomes coordinate different cell death processes remains unclear. Here, we identify the crucial role of MST1/2 in inflammasome-triggered cell death. Macrophages proteolytically convert full-length MST1/2 into the MST1/2 N-terminal fragments (MST1/2-NT) when the NLRC4 inflammasome detects flagellin from the pathogenic bacterium, Legionella pneumophila . Activation of the NLRP3 inflammasome by the damage-associated molecular pattern, extracellular ATP, also produces MST1/2-NT. Caspase-1, the protease activated by these inflammasomes, directly cleaves MST1/2, and blockage of caspase-1 inhibits MST1/2-NT production in macrophages challenged with L. pneumophila . Importantly, MST1/2-NT production is critical for macrophages to trigger a set of death processes associated with apoptosis upon inflammasome activation and knocking out Mst1/2 causes dysregulated gasdermin protein cleavage for pyroptotic death. Furthermore, macrophages lacking MST1/2 have increased susceptibility to virulent L. pneumophila, revealing that the Hippo kinases are important restriction factors against the pathogen. These findings demonstrate that proteolytic cleavage of MST1/2 induced by inflammatory stimuli is an immune pathway to regulate programmed cell death in macrophages and uncover a unique link between the tumor-suppressive Hippo kinases and the inflammasomes in innate immunity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Virus budding is a critical step in the replication cycle of enveloped viruses, closely linked to viral spread, disease progression, and clinical outcomes. The budding of many enveloped RNA viruses is facilitated by the hijacking of the host endosomal sorting complex required for transport (ESCRT) proteins through viral late domains. These late domains are essential for progeny virus production and are highly conserved, making the interaction between late domains and host ESCRT proteins a potential target for the development of antiviral therapeutics. In this study, we elucidated the functional role of the conserved YRTL motif within the glycoprotein Gn cytoplasmic tail of Orthohantavirus hantanense (Hantaan virus, HTNV), demonstrating that HTNV production is regulated by the interaction between YRTL and the ESCRT accessory protein ALIX (ALG-2 interacting protein X). Through virtual molecule docking screening, followed by in vitro and in vivo assays, we discovered a novel compound, AN-329, which disrupts the YRTL-ALIX interaction and effectively inhibits infectious HTNV production, as well as Crimean-Congo hemorrhagic fever virus (CCHFV) and Rift Valley fever virus (RVFV) VLP release. This makes AN-329 a promising therapeutic candidate for reducing viral dissemination. Given that YRTL is conserved across many hantaviruses, our findings may serve as a prototype for the development of broad-spectrum antiviral drugs., (© 2025 Wiley Periodicals LLC.)

Proteins' flexibility is a feature in communicating changes in cell signaling instigated by binding with secondary messengers, such as calcium ions, associated with the coordination of muscle contraction, neurotransmitter release, and gene expression. When binding with the disordered parts of a protein, calcium ions must balance their charge states with the shape of calcium-binding proteins and their versatile pool of partners depending on the circumstances they transmit. Accurately determining the ionic charges of those ions is essential for understanding their role in such processes. However, it is unclear whether the limited experimental data available can be effectively used to train models to accurately predict the charges of calcium-binding protein variants. Here, we developed a chemistry-informed, machine-learning algorithm that implements a game theoretic approach to explain the output of a machine-learning model without the prerequisite of an excessively large database for high-performance prediction of atomic charges. We used the ab initio electronic structure data representing calcium ions and the structures of the disordered segments of calcium-binding peptides with surrounding water molecules to train several explainable models. Network theory was used to extract the topological features of atomic interactions in the structurally complex data dictated by the coordination chemistry of a calcium ion, a potent indicator of its charge state in protein. Our design created a computational tool of Ca X ML, which provided a framework of explainable machine learning model to annotate ionic charges of calcium ions in calcium-binding proteins in response to the chemical changes in an environment. Our framework will provide new insights into protein design for engineering functionality based on the limited size of scientific data in a genome space., (© 2025 Battelle Memorial Institute and The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

A subset of major depressive disorder (MDD) is characterized by immune system dysfunction, but the intracellular origin of these immune changes remains unclear. Here we tested the hypothesis that abnormalities in endoplasmic reticulum (ER) stress, inflammasome activity and mitochondrial biogenesis contribute to the development of systemic inflammation in MDD. RT-qPCR was used to measure mRNA expression of key organellar genes from peripheral blood mononuclear cells (PBMCs) isolated from 186 MDD and 67 healthy control (HC) subjects. The comparative C T (2 -ΔΔCT ) method was applied to quantify mRNA expression using GAPDH as the reference gene. After controlling for age, sex, BMI, and medication status using linear regression models, expression of the inflammasome (NLRC4 and NLRP3) and the ER stress (XBP1u, XBP1s, and ATF4) genes was found to be significantly increased in the MDD versus the HC group. Sensitivity analyses excluding covariates yielded similar results. After excluding outliers, expression of the inflammasome genes was no longer statistically significant but expression of the ER stress genes (XBP1u, XBP1s, and ATF4) remained significant and the mitochondrial biogenesis gene, MFN2, was significantly increased in the MDD group. NLRC4 and MFN2 were positively correlated with serum C-reactive protein concentrations, while ASC trended significant. The altered expression of inflammasome activation, ER stress, and mitochondrial biogenesis pathway components suggest that dysfunction of these organelles may play a role in the pathogenesis of MDD., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: All methods were performed in accordance with the relevant guidelines and regulations. Approval for the Tulsa-1000 (T-1000) study at the Laureate Institute for Brain Research in Tulsa, OK, was obtained from the Western Institutional Review Board screening protocol #20101611. All participants provided written informed consent., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: This study aimed to investigate the impact of aldosterone on calcification in murine vascular smooth muscle cells (VSMCs) via the allograft inflammatory factor-1 (AIF-1)/Wnt/β-catenin signaling pathway., Methods: Mouse VSMCs were cultured in vitro, and calcification was induced by treatment with 100 nM aldosterone. The level of calcification in mouse VSMCs was evaluated using colorimetric assays to assess ALP activity and qRT-PCR to identify the expression of calcification-related markers, such as Runx2, α-SMA, OCN, and ALP mRNA. Western blot analysis was performed to determine the protein expression levels associated with the Wnt/β-catenin pathway (LRP6, p-LRP6, GSK3β, p-GSK3β, β-catenin) and AIF-1. Plasmid transfection techniques were utilized to either knock down or overexpress AIF-1, and the subsequent alterations in these markers were observed., Results: (1) Compared to the control group, the aldosterone treatment group with exhibited a significant increase in ALP. Concurrently, Runx2, OCN, and ALP mRNA levels increased, as did LRP6, p-LRP6, GSK3β, p-GSK3β, β-catenin, and AIF-1 protein levels. Additionally, a significant decrease in the expression of α-SMA mRNA was observed (P < 0.05). (2) The aldosterone + oe-AIF-1 group showed significant increases in ALP activity compared to the aldosterone + oe-NC group, whereas the aldosterone + sh-AIF-1 group showed significant decreases (P < 0.05). (3) The aldosterone + oe-AIF-1 group exhibited significantly upregulated expression of AIF-1, p-LRP6/LRP6, p-GSK3β/GSK3β, and β-catenin proteins relative to the aldosterone + oe-NC group (P < 0.05). This was concurrent with increased mRNA expression of Runx2, OCN, and ALP, and decreased α-SMA mRNA expression (P < 0.05)., Conclusion: Aldosterone affects the calcification process in mouse VSMCs, and the activation of the AIF-1/Wnt/β-catenin signaling pathway is the mechanism behind its action., Competing Interests: Declarations. Conflicts of interest: The authors report there are no competing interests to declare., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Autosomal recessive spinocerebellar ataxias (SCARs) are one of the most common neurodegenerative diseases characterized by progressive ataxia. Although SCARs are known to be caused by mutations in multiple genes, there are still many cases that go undiagnosed or are misdiagnosed. In this study, we presented a SCAR patient, and identified a probable novel pathogenic mutation (c.1A>G, p.M1V) in the AFG3L2 start codon. The proband's genotype included heterozygous mutations of the compound AFG3L2 (p.[M1V]; [R632X] (c.[1A>G]; [1894.C>T])), which were inherited from the father (c.1A>G, p.M1V) and mother (c.1894C>T, p.R632X). Functional studies performed on hiPSCs (human induced pluripotent stem cells) generated from the patients and HEK293T cells showed that the mutations impair mitochondrial function and the unbalanced expression of AFG3L2 mRNA and protein levels. Furthermore, this novel mutation resulted in the degradation of the protein and the reduction of the stability of the AFG3L2 protein, and MCU (mitochondrial calcium uniporter) complex mediated Ca 2+ overload., Competing Interests: Compliance and ethics. The authors declare that they have no conflict of interest., (© 2024. Science China Press.)

Objective: To investigate the effects and mechanisms of miRNA 221 on myocardial ischemia/reperfusion injury (MIRI) in mice through the regulation of phospholamban (PLB) expression., Methods: The MIRI mouse model was created and mice were divided into sham, MIRI, MIRI+ 221, and MIRI+ scr groups, with miRNA 221 overexpression induced in the myocardium of MIRI mice by targeted myocardial injection. Quantitative RT-PCR analysis was performed to observe the variation in miRNA 221, PLB, SERCA2, RYR2, NCX1, Cyt C and caspase 3 mRNA levels in myocardium, while Western blot assessed the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), SERCA2, RYR2, NCX1, Cyt C and caspase 3 proteins. Changes in the structural integrity of the mouse heart were identified with HE and MASSON staining, while TUNEL staining was used to evaluate the TUNEL-positive cells of cardiomyocytes. Changes in myocardium calcium concentration were detected with reagent kits and the targeting interaction between miRNA 221 and PLB was evaluated using a luciferase reporter assay., Results: In the myocardium of MIRI mice, miRNA 221 level was significantly reduced, while the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), and apoptosis-related genes caspase 3, and Cyt C were increased markedly, as well as calcium levels in myocardium. Following the overexpression of miRNA 221 in myocardium, there was a marked alleviation of myocardial injury and cardiomyocyte apoptosis and necrosis, significant enhancement of left ventricular systolic function, and marked decrease in the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), caspase 3 and Cyt C, as well as a significant decrease in total calcium levels in myocardium., Conclusions: miRNA 221 can alleviate myocardial injury in mouse myocardial ischemia/reperfusion by suppressing the expression of PLB, thus reducing calcium overload in myocardium., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Li 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.)

Collagen, a major component of the extracellular matrix, is crucial for the structural integrity of the Caenorhabditis elegans cuticle. While several proteins involved in collagen biosynthesis have been identified, the complete regulatory network remains unclear. This study investigates the role of CALU-1, an ER-resident calcium-binding protein, in cuticle collagen formation and maintenance. We employed genetic analyses, including the generation of single and double mutants, scanning electron microscopy, and transcriptome profiling to characterize CALU-1 function. Our results demonstrate that CALU-1 is essential for proper cuticle structure, including annuli, furrows, and alae formation. Synthetic lethality was observed between calu-1 and dpy-18 (encoding a prolyl 4-hydroxylase subunit) mutations, while double mutants of calu-1 with peptidyl-prolyl cis-trans isomerase (PPIase) genes exhibited exacerbated phenotypes. CALU-1 deficiency led to altered collagen stability, increased cuticle permeability, and differential expression of stress response genes similar to collagen mutants. We conclude that CALU-1 plays a critical role in regulating collagen biosynthesis, possibly by modulating the ER environment to optimize the function of collagen-modifying enzymes. These findings provide new insights into the complex regulation of extracellular matrix formation in C. elegans, with potential implications for understanding related processes in other organisms., Competing Interests: Declarations. Conflict of interests: The authors declare no conflict of interest. Consent for publication: All authors consented to publish the manuscript., (© 2025. The Author(s).)

The brain presents various structural and functional sex differences, for which multiple factors are attributed: genetic, epigenetic, metabolic, and hormonal. While biological sex is determined by both sex chromosomes and sex hormones, little is known about how these two factors interact to establish this dimorphism. Sex differences in the brain also affect its resident immune cells, microglia, which actively survey the brain parenchyma and interact with sex hormones throughout life. However, microglial differences in density and distribution, morphology and ultrastructural patterns in physiological conditions during adulthood are largely unknown. Here, we investigated these aforementioned properties of microglia using the Four Core Genotypes (FCG) model, which allows for an independent assessment of gonadal hormones and sex chromosomal effects in four conditions: FCG XX and Tg XY - (both ovaries); Tg XX Sry and Tg XY Sry (both testes). We also compared the FCG results with XX and XY wild-type (WT) mice. In adult mice, we focused our investigation on the ventral hippocampus across different layers: CA1 stratum radiatum (Rad) and CA1 stratum lacunosum-moleculare (LMol), as well as the dentate gyrus polymorphic layer (PoDG). Double immunostaining for Iba1 and TMEM119 revealed that microglial density is influenced by both sex chromosomes and sex hormones. We show in the Rad and LMol that microglia are denser in FCG XX compared to Tg XY Sry mice, however, microglia were densest in WT XX mice. In the PoDG, ovarian animals had increased microglial density compared to testes animals. Additionally, microglial morphology was modulated by a complex interaction between hormones and chromosomes, affecting both their cellular soma and arborization across the hippocampal layers. Moreover, ultrastructural analysis showed that microglia in WT animals make overall more contacts with pre- and post-synaptic elements than in FCG animals. Lastly, microglial markers of cellular stress, including mitochondrion elongation, and dilation of the endoplasmic reticulum and Golgi apparatus, were mostly chromosomally driven. Overall, we characterized different aspects of microglial properties during normal physiological conditions that were found to be shaped by sex chromosomes and sex hormones, shading more light onto how sex differences affect the brain immunity at steady-state., Competing Interests: Declarations. Ethics approval: All animal experiments and procedures were approved by the Université Laval (2021-830 and 2023-1372) and the University of Victoria (AUP 2020-013) animal care committees, strictly following the recommendations from the Canadian Council on Animal Care. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

An actin-binding protein, known as Calponin 3 (CNN3), modulates the remodeling of the actin cytoskeleton, a fundamental process for the maintenance of skeletal muscle homeostasis. Although the roles of CNN3 in actin remodeling have been established, its biological significance in myoblast differentiation remains largely unknown. This study investigated the functional significance of CNN3 in myogenic differentiation, along with its effects on actin remodeling and mechanosensitive signaling in C2C12 myoblasts. CNN3 knockdown led to a marked increase in filamentous actin, which promoted the nuclear localization of Yes-associated protein 1 (YAP1), a mechanosensitive transcriptional coactivator required for response to the mechanical cues that drive cell proliferation. Subsequently, CNN3 depletion enhanced myoblast proliferation by upregulating the expression of the YAP1 target genes related to cell cycle progression, such as cyclin B1, cyclin D1, and PCNA. According to a flow cytometry analysis, CNN3-deficient cells displayed higher S and G2/M phase fractions, which concurred with elevated proliferation rates. Furthermore, CNN3 knockdown impaired myogenic differentiation, as evidenced by reduced levels of MyoD, MyoG, and MyHC, key markers of myogenic commitment and maturation, and immunocytochemistry showed that myotube formation was diminished in CNN3-suppressed cells, which was supported by lower differentiation and fusion indices. These findings reveal that CNN3 is essential for myogenic differentiation, playing a key role in regulating actin remodeling and cellular localization of YAP1 to orchestrate the proliferation and differentiation in myogenic progenitor cells. This study highlights CNN3 as a critical regulator of skeletal myogenesis and suggests its therapeutic potential as a target for muscle atrophy and related disorders.

Objective: To investigate the clinical features and genetic variants associated with Multiple mitochondrial dysfunction syndrome (MMDS) type 3 in two children., Methods: Two children diagnosed with MMDS type 3 at Zhuhai Maternal and Child Health Care Hospital in January 2021 were selected for this study. A retrospective analysis of their clinical data was carried out. Whole exome sequencing was conducted on the two children and their parents, followed by Sanger sequencing for candidate variants and bioinformatic analysis. Both children received comprehensive rehabilitative therapy and were followed up for 3 years. This study was approved by the Ethics Committee of Zhuhai Maternal and Child Health Hospital (Ethics No. 202380)., Results: The two MMDS type 3 children were monozygotic twin girls, aged 9 months, presenting with developmental regression, pyramidal signs, and other clinical manifestations. Cranial MRI revealed widespread abnormal signals and vacuolar changes in the white matter. Whole exome sequencing revealed that both children had harbored compound heterozygous variants of the IBA57 gene, namely c.286T>C (p.Tyr96His) and c.307C>T (p.Gln103Ter). Sanger sequencing confirmed that these variants were inherited from their father and mother, respectively. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, both variants were classified as pathogenic (PM2&#95;Supporting+PM3&#95;Very Strong+PP3&#95;Moderate; PVS1+PM2&#95;Supporting+PM3). After treatment with vitamins, levocarnitine, ATP, coenzyme Q10, and other drugs, both children showed partial recovery of neurodevelopmental regression, with improvement in feeding and sleep. Over the 3-year follow-up, there was slow but progressive improvement in motor, language, and cognitive development., Conclusion: The compound heterozygous variants c.286T>C (p.Tyr96His) and c.307C>T (p.Gln103Ter) of the IBA57 gene probably underlay the MMDS type 3 in the twin pair. Clinicians should be vigilant about the possibility of MMDS type 3 in children with neurodevelopmental regression and early cranial MRI findings indicating widespread white matter abnormalities with vacuolar changes, as these may be indicative of IBA57 gene variants.

Ferroptosis is a newly identified programmed cell death induced by iron-driven lipid peroxidation and implicated as a potential approach for tumor treatment. However, emerging evidence indicates that hepatocellular carcinoma (HCC) cells are generally resistant to ferroptosis and the underlying molecular mechanism is poorly understood. Here, our study confirms that S100 calcium binding protein P (S100P), which is significantly up-regulated in ferroptosis-resistant HCC cells, efficiently inhibits ferroptosis. Mechanistically, S100P facilitates lysosomal degradation of acetyl-CoA carboxylase alpha (ACC1), which is indispensable for de novo biosynthesis of lipids. Loss of S100P elevates the expression of ACC1 and promotes ferroptotic sensitivity of HCC cells. S100P-mediated ACC1 degradation relies on RAB5C, which directs ACC1 to lysosome via P62-dependent selective autophagy. Knockdown of RAB5C or P62 abrogates S100P-induced lysosomal degradation of ACC1 and restores resistance of HCC cells to ferroptosis. Our work reveals an alternative anti-ferroptosis pathway and suggests S100P as a promising druggable target for ferroptosis-related therapy of HCC., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Glycans are known to be fundamental for many cellular and physiological functions. Congenital disorders of glycosylation (CDG) currently encompassing over 160 subtypes, are characterized by glycan synthesis and/or processing defects. Despite the increasing number of CDG patients, therapeutic options remain very limited as our knowledge on glycan synthesis is fragmented. The emergence of CDG resulting from defects in ER/ Golgi homeostasis makes this even more difficult. SLC10A7 belongs to the SLC10 protein family, known as bile acid and steroid transport family, exhibiting a unique structure. It shows a ubiquitous expression and is linked to negative calcium regulation in cells. The mechanisms by which SLC10A7 deficiency leads to Golgi glycosylation abnormalities are unknown. The present study identifies major O-glycosylation defects in both SLC10A7 KO HAP1 cells and SLC10A7-CDG patient fibroblasts and reveals an increased ER and Golgi calcium contents. We also show that the abundance of COSMC and C1GALT1 is altered in SLC10A7-CDG patient cells, as well as the subcellular Golgi localization of the Ca 2+ -binding Cab45 protein. Finally, we demonstrate that supraphysiological manganese supplementation suppresses the deficient electrophoretic mobility of TGN46 by an aberrant transfer of GalNAc residues, and reveal COSMC Mn 2+ sensitivity. These findings provide novel insights into the mechanisms of Golgi glycosylation defects in SLC10A7-deficient cells. They show that SLC10A7 is a key Golgi transmembrane protein maintaining the tight regulation of Ca 2+ homeostasis in the ER and Golgi compartments, both essential for glycosylation., Competing Interests: Declarations. Conflict of 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. Ethics approval and consent to participate: The study was conducted in accordance with the Declaration of Helsinki. All subjects and guardians provided written informed consent for the analysis of biological samples, following the approval of local ethics committees. Consent for publication: All authors read and approved the final manuscript and gave consent for the publication of the manuscript., (© 2025. The Author(s).)

Background: Congenital anomalies (CAs) encompass a wide spectrum of structural and functional abnormalities during fetal development, commonly presenting at birth. Identifying the cause of CA is essential for accurate diagnosis and treatment. Using a target-gene approach, genetic variants could be found in certain CA patients. However, some patients were genetically undiagnosed; therefore, it is imperative to identify the causative variants from whole genome sequence (WGS) data of these patients., Results: An in-house pipeline utilizing DRAGEN-GATK-Hail was established for trio-based WGS data analysis (n = 18 undiagnosed CA patients and their parents) and thirty-five candidate variants, including SNV/Indel, CNV, and SV were identified. Among them, 10 variants of seven coding genes were selected as possible causal variants by variant pathogenicity, genotype-phenotype analysis, and a multidisciplinary team. Finally, functional validation of six genes including RYR3, NRXN1, FREM2, CSMD1, RARS1, and NOTCH1, revealed various phenotypes in zebrafish models that aligned with those observed in each patient. In addition to the above findings, eleven diagnostic variants initially discovered in a targeted-gene analysis from a previous study were also identified as diagnostic variants and the in-house pipeline demonstrated a significant advantage in accurately and efficiently identifying de novo variants (DNVs), compound heterozygous (CH), and homozygous variants., Conclusions: Taken together, the in-house pipeline established in this study provides a highly valuable diagnostic tool for the identification of potential candidate variants in patients with CA. Further research into the molecular mechanisms related to the development of CAs could shed light on the functional aspects of these genetic variations and contribute to the development of therapeutic drugs., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

De-regulated protein expression contributes to tumor growth and progression in medulloblastoma (MB), the most common malignant brain tumor in children. MB is associated with impaired differentiation of specific neural progenitors, suggesting that the deregulation of proteins involved in neural physiology could contribute to the transformed phenotype in MB. Calsynthenin 1 (CLSTN1) is a neuronal protein involved in cell-cell interaction, vesicle trafficking, and synaptic signaling. We previously identified CLSTN1 as a putative target of the pro-invasive kinase MAP4K4, which we found to reduce CLSTN1 surface expression. Herein, we explored the expression and functional significance of CLSTN1 in MB. We found that CLSTN1 expression is decreased in primary MB tumors compared to tumor-free cerebellum or brain tissues. CLSTN1 is expressed in laboratory-established MB cell lines, where it localized to the plasma membrane, intracellular vesicular structures, and regions of cell-cell contact. The reduction of CLSTN1 expression significantly increased growth factor-driven invasiveness. Pharmacological inhibition of pro-migratory MAP4 kinases caused increased CLSTN1 expression and CLSTN1 accumulation in cell-cell contacts. Co-culture of tumor cells with astrocytes increased CLSTN1 localization in cell-cell contacts, which was further enhanced by MAP4K inhibition. Our study revealed a repressive function of CLSTN1 in growth-factor-driven invasiveness in MB, identified MAP4 kinases as repressors of CLSTN1 recruitment to cell-cell contacts, and points towards CLSTN1 implication in the kinase-controlled regulation of tumor-microenvironment interaction., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Protein secretion is an essential process that drives cell growth and communication. Enrichment of soluble secretory proteins into ER-derived transport carriers occurs via transmembrane cargo receptors that connect lumenal cargo to the cytosolic COPII coat. Here, we find that the cargo receptor, SURF4, recruits different SEC24 cargo adaptor paralogs of the COPII coat to export different cargoes. The secreted protease, PCSK9, requires both SURF4 and a co-receptor, TMED10, for export via SEC24A. In contrast, secretion of Cab45 and NUCB1 requires SEC24C/D. We further show that ER export signals of Cab45 and NUCB1 bind co-translationally to SURF4 via a lumenal pocket, contrasting prevailing models of receptor engagement only upon protein folding/maturation. Bioinformatics analyses suggest that strong SURF4-binding motifs are features of proteases, receptor-binding ligands, and Ca2+-binding proteins. We propose that certain classes of proteins are fast-tracked for rapid export to protect the health of the ER lumen., (© 2024 MRC Laboratory of Molecular Biology.)

The Notch signaling pathway uses families of ligands and receptors to transmit signals to nearby cells. These components are expressed in diverse combinations in different cell types, interact in a many-to-many fashion, both within the same cell (in cis) and between cells (in trans), and their interactions are modulated by Fringe glycosyltransferases. A fundamental question is how the strength of Notch signaling depends on which pathway components are expressed, at what levels, and in which cells. Here, we used a quantitative, bottom-up, cell-based approach to systematically characterize trans-activation, cis-inhibition, and cis-activation signaling efficiencies across a range of ligand and Fringe expression levels in Chinese hamster and mouse cell lines. Each ligand (Dll1, Dll4, Jag1, and Jag2) and receptor variant (Notch1 and Notch2) analyzed here exhibited a unique profile of interactions, Fringe dependence, and signaling outcomes. All four ligands were able to bind receptors in cis and in trans, and all ligands trans-activated both receptors, although Jag1-Notch1 signaling was substantially weaker than other ligand-receptor combinations. Cis-interactions were predominantly inhibitory, with the exception of the Dll1- and Dll4-Notch2 pairs, which exhibited cis-activation stronger than trans-activation. Lfng strengthened Delta-mediated trans-activation and weakened Jagged-mediated trans-activation for both receptors. Finally, cis-ligands showed diverse cis-inhibition strengths, which depended on the identity of the trans-ligand as well as the receptor. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway., Competing Interests: RK, LS, ME No competing interests declared, (© 2023, Kuintzle et al.)

Background: Vitamin K-dependent γ-glutamic acid carboxylation (Gla) proteins are calcium-binding and membrane-associated, participating in coagulation, bone turnover, and cancer biology. The molecular function of transmembrane proline-rich Gla proteins (PRRGs) remains unexplored., Methods: Analysis of pancreatic ductal adenocarcinoma (PDAC) datasets, including transcription profiles, clinical data, and tissue microarrays, was conducted to evaluate PRRG1 expression and its clinical relevance. PDAC cell lines with overexpressed, knockdown, and mutated PRRG1 were developed to study biological functions and pathways using RNA-seq, co-immunoprecipitation with mass spectrometry, Western blotting, and immunofluorescence. In vivo xenograft and orthotopic models assessed PRRG1's impact on PDAC progression, with and without warfarin treatment., Results: PRRG1 was significantly upregulated in PDAC compared to normal pancreas, correlating with poorer patient survival. PRRG1 knockdown reduced PDAC cell proliferation, anchorage-independent growth in vitro, and tumor growth in vivo. PRRG1 localized at the plasma membrane, interacted with the HECT E3 ligase NEDD4 via the C-terminal PPXY motif, and promoted NEDD4 self-ubiquitination, reducing its protein levels. PRRG1 knockdown elevated NEDD4, destabilizing the oncoprotein KRAS and receptor EGFR, and attenuating downstream signaling and macropinocytosis under nutrient deprivation. The vitamin K-dependent Gla modification of PRRG1 was crucial for its membrane localization and pro-tumorigenic effects, and was inhibited by low-dose warfarin, a clinical vitamin K antagonist., Conclusions: This study identifies PRRG1 as a key regulator of pro-tumorigenic signaling in PDAC, suggesting the potential of repurposing the anticoagulant warfarin as a therapeutic strategy., Key Points: PRRG1 is identified as the transmembrane Gla protein mediating PDAC malignancy. PRRG1 recruits and induces self-ubiquitination of membrane-anchoring E3 ligase NEDD4. PRRG1 exerts a protective role toward KRAS and EGFR by inhibiting NEDD4. The anticoagulant warfarin can be utilized to inhibit PRRG1 and PDAC advancement., (© 2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Calcium ions (Ca 2+ ) are important second messengers and are known to participate in cold signal transduction. In the current study, we characterized a Ca 2+ -binding protein gene, VamCP1, from the extremely cold-tolerant grape species Vitis amurensis. VamCP1 expression varied among organs but was highest in leaves following cold treatment, peaking 24 h after treatment onset. VamCP1 was found to localize to the plasma membrane and nucleus and the gene showed transcriptional autoactivation activity. Overexpression of VamCP1 in Arabidopsis thaliana and grapevine (V. vinifera) resulted in transgenic plants that were more tolerant to cold stress than the wild type. This correlated with reduced accumulation of reactive oxygen species (ROS), elevated activity of antioxidant enzymes and proline content, as well as lower levels of malondialdehyde and electrolyte leakage. Additionally, the expression of genes related to cold tolerance, including C-repeat binding factors (CBF) and cold-regulated (COR) genes, was higher in the transgenic lines. Taken together, our results indicate that overexpression of VamCP1 enhanced cold tolerance in plants by promoting the upregulation of genes related to cold tolerance and scavenging of excessive ROS. These findings provide a foundation for the molecular breeding of cold-tolerant grapevine., (© 2025 Scandinavian Plant Physiology Society.)

The activation of the human interferon-inducible protein X (IFIX) isoform is associated with maintaining a stable cytoskeleton and inhibiting epithelial-mesenchymal transition (EMT). However, the mechanisms and pathways underlying IFIX-mediated oncogenesis are not well understood. In this study, we investigated the effects of IFIX overexpression and knockdown in CAL-27 and SCC-25 oral squamous cell carcinoma (OSCC) cells. We observed significant variations in the expression of E-cadherin, N-cadherin, vimentin and Snail, as well as changes in wingless/integrated (Wnt) signalling. Our results indicated a strong correlation between IFIX and EMT, as evidenced by quantitative reverse-transcription PCR and Western blotting, which revealed that Wnt3a and Wnt4 pathway components were regulated in IFIX-overexpressing or knockdown cells, with naked cuticle 2 (NKD2) showing the strongest positive correlation. Both IFIX overexpression and knockdown modulated NKD2 expression. NKD2 silencing mimicked the phenotypic effects of IFIX knockdown, inhibiting E-cadherin expression and increasing N-cadherin, Snail and vimentin expression. Additionally, silencing NKD2 restored the anticarcinogenic phenotype associated with IFIX overexpression, affecting cell proliferation, invasion and migration. These findings provide mechanistic insights into the antioncogenic effects of IFIX in OSCC, involving the inhibition of Wnt signalling through NKD2, which leads to cancer-inhibiting phenotypic effects, including restricted EMT., (© 2025 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Autism spectrum disorder (ASD) is a set of heterogeneous neurodevelopmental conditions, with a highly diverse genetic hereditary component, including altered neuronal circuits, that has an impact on communication skills and behaviours of the affected individuals. Beside the recognised role of neuronal alterations, perturbations of microglia and the associated neuroinflammatory processes have emerged as credible contributors to aetiology and physiopathology of ASD. Mutations in NRXN1, a member of the neurexin family of cell-surface receptors that bind neuroligin, have been associated to ASD. NRXN1 is known to be expressed by neurons where it facilitates synaptic contacts, but it has also been identified in glial cells including microglia. Asserting the impact of ASD-related genes on neuronal versus microglia functions has been challenging. Here, we present an ASD subject-derived induced pluripotent stem cells (iPSC)-based in vitro system to characterise the effects of the ASD-associated NRXN1 gene deletion on neurons and microglia, as well as on the ability of microglia to support neuronal circuit formation and function. Using this approach, we demonstrated that NRXN1 deletion, impacting on the expression of the alpha isoform (NRXN1α), in microglia leads to microglial alterations and release of IL6, a pro-inflammatory interleukin associated with ASD. Moreover, microglia bearing the NRXN1α-deletion, lost the ability to support the formation of functional neuronal networks. The use of recombinant IL6 protein on control microglia-neuron co-cultures or neutralizing antibody to IL6 on their NRXN1α-deficient counterparts, supported a direct contribution of IL6 to the observed neuronal phenotype. Altogether, our data suggest that, in addition to neurons, microglia are also negatively affected by NRXN1α-deletion, and this significantly contributes to the observed neuronal circuit aberrations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: B.J. is co-founder of CERVO Therapeutics AB. S.C. and S.S. are co-founders of NorthernLight Diagnostics AB. The other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout ( Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 ( Smoc1 ) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 ( Pax7 ) and myogenic factor 5 ( Myf5 ). After short-interfering RNA-mediated knocking down of Smoc1 , the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent., (Copyright ©The Author(s)(2024).)

Communication of gut hormones with the central nervous system is important to regulate systemic glucose homeostasis, but the precise underlying mechanism involved remain little understood. Nesfatin-1, encoded by nucleobindin-2 (NUCB2), a potent anorexigenic peptide hormone, was found to be released from the gastrointestinal tract, but its specific function in this context remains unclear. Herein, we found that gut nesfatin-1 can sense nutrients such as glucose and lipids and subsequently decreases hepatic glucose production. Nesfatin-1 infusion in the small intestine of NUCB2-knockout rats reduced hepatic glucose production via a gut - brain - liver circuit. Mechanistically, NUCB2/nesfatin-1 interacted directly with melanocortin 4 receptor (MC4R) through its H-F-R domain and increased cyclic adenosine monophosphate (cAMP) levels and glucagon-like peptide 1 (GLP-1) secretion in the intestinal epithelium, thus inhibiting hepatic glucose production. The intestinal nesfatin-1 -MC4R-cAMP-GLP-1 pathway and systemic gut-brain communication are required for nesfatin-1 - mediated regulation of liver energy metabolism. These findings reveal a novel mechanism of hepatic glucose production control by gut hormones through the central nervous system., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Research underscores the urgent need for technological innovations to treat lung tissue damage from viral infections and the lasting impact of COVID-19. Our study demonstrates the effectiveness of recombinant human NV1 protein in promoting a pro-healing extracellular matrix that regulates homeostasis in response to excessive tissue reactions caused by infection and injury. NV1 achieves this by calibrating multiple biological mechanisms, including reducing hyperinflammatory cytokine levels (e.g., IFN-γ, TNF-α, IL-10, and IP-10), enhancing the production of proteins involved in viral inactivation and clearance through endocytosis and phagocytosis (e.g., IL-9, IL-1α), regulating pro-clotting and thrombolytic pathways (e.g., downregulates SERPINE 1 and I-TAC during Th1-mediated inflammation), maintaining cell survival under hypoxic conditions via HIF-1α regulation through the M3K5-JNK-AP-1 and TSC2-mTOR pathways, and promoting blood vessel formation. Our findings reveal NV1 as a potential therapeutic candidate for treating severe lung injuries caused by inflammatory and hypoxic conditions from viral infections and related diseases., Competing Interests: Declaration of competing interest C.C. is sole inventor on NELL1 patents exclusively licensed from UT-Battelle LLC (Oak Ridge, TN, USA) and NV1 patents owned by NellOne Therapeutics Inc. (NTI). C.C. and S.E. are co-inventors on NTI-owned patents for healing muscle atrophy and viral-induced tissue damage with NELL1 and NV1. C.C. is co-founder and CSO, and W.M. is CEO of NTI. The remaining authors declare no competing financial interests., (Published by Elsevier Inc.)

Citrin belongs to the SLC25 transport protein family found mostly in inner mitochondrial membranes. The family prototype, the ADP-ATP carrier, delivers ATP made inside mitochondria to the cellular cytoplasm and returns ADP to the mitochondrion for resynthesis of ATP. In pre-genomic 1981, I noticed that the protein sequence of the bovine ADP-ATP carrier consists of three related sequences, each containing two transmembrane α-helices traveling in opposite senses. Colleagues and I demonstrated that two other mitochondrial carriers had similar features. From emergent genomic sequences, it became apparent that they represented a large family of transport proteins with the same characteristic threefold repeats. The human genome encodes 53 members, but the functions of many were unknown. So, colleagues and I determined how to make these proteins in Escherichia coli and introduce them into liposomes to allow exploration of their transport functions. The 27 human family members to have been thus identified include citrin and the closely related protein aralar. Both exchange aspartate from the mitochondrial matrix for cytosolic glutamate plus a proton. Citrin is expressed predominantly in liver and non-excitable tissues, whereas aralar is the dominant form in the brain. Each has a membrane extrinsic N-terminal Ca 2+ -binding domain, a transport domain, and a C-terminal amphipathic α-helix. Human mutations in citrin impair the urea cycle, malate-aspartate shuttle, gluconeogenesis, amino acid breakdown, and energy metabolism leading to citrin deficiency. Currently, the complex etiology of this condition is poorly understood and new knowledge would help to improve diagnosis, therapies, and finding a cure. My aims are to seek a basic understanding of the etiology of citrin deficiency and to use that knowledge in improving diagnostic procedures and in developing new treatments and a cure., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Age-related bone loss in mice is associated with senescent cell accumulation and reduced bone formation by osteoblasts. Matrix Gla protein (MGP), secreted by osteoblasts, is pivotal in regulating the bone extracellular matrix mineralization. Previous research has demonstrated that Mgp null mice exhibit osteopenia and fractures, and ultimately die prematurely. To elucidate the mechanisms underlying MGP's role of MGP in bone metabolism, we generated osteoblast-specific Mgp knockout (Mgp cKO) mice by crossing Mgp fl/fl mice with Bglap-Cre mice. The study revealed that in 3-month-old Mgp cKO male mice, trabecular bone volume decreased, and the senescence marker protein p21 increased. Primary osteoblasts from Mgp cKO mice exhibited markers of DNA damage and senescence, such as increased γH2AX foci, p21, and senescence-associated β-galactosidase staining, as well as attenuated cellular proliferation and osteogenic differentiation abilities. In addition, bone marrow stromal cells' colony formation and spontaneous osteogenic ability were impaired in Mgp cKO mice, whereas osteoclastogenesis was enhanced. In vitro treatment with recombinant human MGP promotes osteogenesis in osteoblasts derived from Mgp cKO mice via the PI3K-AKT signaling pathway. Thus, our results suggest that MGP is protective by suppressing osteoblast senescence, offering new insights into potential therapeutic strategies for age-related osteoporosis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Impaired cardiomyocyte Ca 2+ handling is a central hallmark of heart failure (HF), which causes contractile dysfunction and arrhythmias. However, the underlying molecular mechanisms and the precise contribution of defects in Ca 2+ -cycling regulation in the development of HF are still not completely resolved. Here, we used transgenic mice that express a human mutation in the cardiomyocyte Ca 2+ -regulator phospholamban (PLN R9C -tg) causing severe HF due to a reduction in Ca 2+ reuptake into the sarco(endo)plasmic reticulum (SR). PLN R9C -induced HF is a rapidly progressing condition characterized by prominent Ca 2+ cycling and relaxation defects and premature death of mutation carriers. We found that endoplasmic reticulum (ER) and mitochondrial function are affected even before transition to overt HF. Early correction of aberrant Ca 2+ cycling by cardiac expression of the Raf kinase inhibitor protein (RKIP), an endogenous activator of β-adrenoceptors (βAR), delayed the cellular alterations, functional failure and prolonged lifespan. Our study highlights the importance of early and persistent correction of Ca 2 + dynamics, not only for excitation/contraction coupling, but also for the prevention of rather irreparable events on cardiac energetics and ER stress adaptations. The latter may even impede with later onset of Ca 2+ -related therapeutic interventions and should gain more focus for HF treatment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kristina Lorenz, Joachim Schmitt reports financial support was provided by German Research Foundation. Kristina Lorenz reports financial support was provided by Federal Ministry of Education and Research Bonn Office. Kristina Lorenz reports financial support was provided by Ministry of Culture and Science of the State of North Rhine-Westphalia. Martina Krueger, Petra Kleinbongard, Albert Sickmann reports financial support was provided by German Research Foundation. Reports a relationship with that includes:. Has patent pending to. If there are other authors, they 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 © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Myocardial infarction (MI) mobilizes macrophages, the central protagonists of tissue repair in the infarcted heart. Although necessary for repair, macrophages also contribute to adverse remodeling and progression to heart failure. In this context, specific targeting of inflammatory macrophage activation may attenuate maladaptive responses and enhance cardiac repair. Allograft inflammatory factor 1 (AIF1) is a macrophage-specific protein expressed in a variety of inflammatory settings, but its function after MI is unknown. Here we identify a maladaptive role for macrophage AIF1 after MI in mice. Mechanistic studies show that AIF1 increases actin remodeling in macrophages to promote reactive oxygen species-dependent activation of hypoxia-inducible factor (HIF)-1α. This directs a switch to glycolytic metabolism to fuel macrophage-mediated inflammation, adverse ventricular remodeling and progression to heart failure. Targeted knockdown of Aif1 using antisense oligonucleotides improved cardiac repair, supporting further exploration of macrophage AIF1 as a therapeutic target after MI., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Delta-like homolog 2 (DLK2) plays a crucial role in adipogenesis, chondrogenic differentiation, and the progression of certain cancers. However, the key roles of DLK2 underlying the progression of hepatocellular carcinoma (HCC) remain ambiguous. In the current study, we demonstrate that DLK2 is upregulated in HCC, significantly correlated with clinicopathological variables and serves as an independent diagnostic marker. Functional assays reveal that DLK2 facilitates malignant progression of HCC in vitro and in vivo models. Mechanistically, DLK2 binds to EGFR resulting in its auto-phosphorylation, which activates NF-κB pathway leading to P65-dependent transcriptional upregulation of PKM2. Furthermore, that elevates both enzyme-dependent and -independent activities of PKM2 contributing to cancer proliferation and metastasis. In summary, our findings demonstrate a novel pro-tumoral role and mechanism of DLK2 in the regulation of HCC malignant progression, suggesting its potential as a clinical diagnostic marker and therapeutic target., (© 2024 Wiley Periodicals LLC.)

The Phenotypic states of vascular smooth muscle cells (SMCs) are essential to understanding vascular pathophysiology. SMCs in vessels generally express a specific set of contractile proteins, but decreased contractile protein expression, indicating a phenotypic shift, is a hallmark of vascular diseases. Recent studies have suggested the relation of abnormally high wall shear stress (WSS) of approximately 20 Pa with the aortic disease pathogenesis. However, due to the lack of appropriate experimental models to assess SMC phenotypic states, the details of the phenotypic shift under high WSS conditions remain unclear. In this study, we developed a coculture model where vascular endothelial cells (ECs) were cocultured with SMCs expressing calponin 1, a contractile protein involved in the phenotypic shift of SMCs. We investigated the effects of a pathologically high WSS condition on the phenotypic states of SMCs. Increased calponin 1 expression was found upon exposure to 20 Pa WSS compared with a physiological 2 Pa condition, whereas the expression of another contractile protein, α-smooth muscle actin (αSMA) remained unchanged. Furthermore, the inhibition of EC-derived nitric oxide (NO), which is associated with endothelial dysfunction in vascular diseases, resulted in a trend of decreasing αSMA and Calponin 1 expression under 20 Pa WSS conditions compared with 2 Pa. Our findings suggest that EC-derived NO under pathologically high WSS conditions may impact the expression of contractile proteins implicated in aortic pathophysiology., 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.)

To encode continuous sound stimuli, the inner hair cell (IHC) ribbon synapses utilize calcium-binding proteins (CaBPs), which reduce the inactivation of their Ca V 1.3 calcium channels. Mutations in the CABP2 gene underlie non-syndromic autosomal recessive hearing loss DFNB93. Besides CaBP2, the structurally related CaBP1 is highly abundant in the IHCs. Here, we investigated how the two CaBPs cooperatively regulate IHC synaptic function. In Cabp1/2 double-knockout mice, we find strongly enhanced Ca V 1.3 inactivation, slowed recovery from inactivation and impaired sustained exocytosis. Already mild IHC activation further reduces the availability of channels to trigger synaptic transmission and may effectively silence synapses. Spontaneous and sound-evoked responses of spiral ganglion neurons in vivo are strikingly reduced and strongly depend on stimulation rates. Transgenic expression of CaBP2 leads to substantial recovery of IHC synaptic function and hearing sensitivity. We conclude that CaBP1 and 2 act together to suppress voltage- and calcium-dependent inactivation of IHC Ca V 1.3 channels in order to support sufficient rate of exocytosis and enable fast, temporally precise and indefatigable sound encoding., Competing Interests: DO, SC, KK, VR, SJ, NS, TP No competing interests declared, (© 2024, Oestreicher et al.)

Homeothermic endotherms defend their body temperature in cold environments using a number of behavioral and physiological mechanisms. Maintaining a stable body temperature primarily requires heat production through shivering or non-shivering thermogenesis (NST). Although the use of NST is well established in mammalian systems, the mechanisms and extent to which NST is used in birds are poorly understood. In mammals, one well-characterized mechanism of NST is through uncoupling of Ca2+ transport from ATP hydrolysis by sarco/endoplasmic reticulum ATPase (SERCA) in the skeletal muscle, which generates heat and may contribute to Ca2+ signaling for fatigue resistance and mitochondrial biogenesis. Two small proteins-sarcolipin (SLN) and phospholamban (PLN)-are known to regulate SERCA in mammals, but recent work shows inconsistent responses of SLN to cold acclimation in birds. In this study, we measured SERCA uncoupling in the pectoralis flight muscle of control (18°C) and cold-acclimated (-8°C) dark-eyed juncos (Junco hyemalis) that exhibited suppressed SLN transcription in the cold. We measured SERCA activity and Ca2+ uptake rates for the first time in cold-acclimated birds and found greater SERCA uncoupling in the muscle of juncos in the cold. However, SERCA uncoupling was not related to SLN or PLN transcription or measures of mitochondrial biogenesis. Nonetheless, SERCA uncoupling reduced an individual's risk of hypothermia in the cold. Therefore, while SERCA uncoupling in the cold could be indicative of NST, it does not appear to be mediated by known regulatory proteins in these birds. These results prompt interesting questions about the significance of SLN and PLN in birds and the role of SERCA uncoupling in response to environmental conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Understanding cell fate regulation in the liver is necessary to advance cell therapies for hepatic disease. Liver progenitor cells (LPCs) contribute to tissue regeneration after severe hepatic injury, yet signals instructing progenitor cell dynamics and fate are largely unknown. Tissue inhibitor of metalloproteinases 1 (TIMP1) and TIMP3 control the sheddases ADAM10 and ADAM17, key for NOTCH activation. Here we uncover the role of the TIMP/ADAM/NOTCH/DLK1 axis in LPC maintenance and cholangiocyte specification. Combined TIMP1/TIMP3 loss in vivo caused abnormal portal triad stoichiometry accompanied by collagen deposits, dysregulated Notch signaling, and increased soluble DLK1. The MIC1-1C3+CD133+CD26- biliary progenitor population was reduced following acute CCl4 or chronic DDC liver injury and in aged TIMP-deficient livers. Single-cell RNA sequencing data interrogation and RNAscope identified portal mesenchymal cells coexpressing ADAM17/DLK1 as enzymatically equipped to process DLK1 and direct LPC differentiation. Specifically, TIMP-deficient biliary fragment-derived organoids displayed increased propensity for cholangiocyte differentiation. ADAM17 inhibition reduced Sox9-mediated cholangiocyte differentiation, prolonging organoid growth and survival, whereas WT organoids treated with soluble DLK1 triggered Sox9 expression and cholangiocyte specification in mouse and patient-derived liver organoids. Thus, metalloproteinase inhibitors regulate instructive signals for biliary cell differentiation and LPC preservation within the portal niche, providing a new basis for cell therapy strategies.

The coordinate regulation of metabolism and epigenetics to establish cell state-specific gene expression patterns during lineage progression is a central aspect of cell differentiation, but the factors that regulate this elaborate interplay are not well-defined. The imprinted Dlk1-Dio3 noncoding RNA (ncRNA) cluster has been associated with metabolism in various progenitor cells, suggesting it functions as a regulator of metabolism and cell state. Here, we directly demonstrate that the Dlk1-Dio3 ncRNA cluster coordinates mitochondrial respiration and chromatin structure to maintain proper cell state. Stable mouse muscle cell lines were generated harboring two distinct deletions in the proximal promoter region, resulting in either greatly upregulated or downregulated expression of the entire Dlk1-Dio3 ncRNA cluster. Both mutant lines displayed impaired muscle differentiation along with dysregulated structural gene expression and abnormalities in mitochondrial respiration. Genome-wide chromatin accessibility and histone methylation patterns were also severely affected in these mutants. Our results strongly suggest that muscle cells are sensitive to Dlk1-Dio3 ncRNA dosage, and that the cluster coordinately regulates metabolic activity and the epigenome to maintain proper cell state in the myogenic lineage., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Objectives: To observe the reparative effects of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation on white matter injury (WMI) in neonatal rats and explore its mechanism through the nuclear factor-kappa B (NF-κB) signaling pathway mediated by microglial cells., Methods: Sprague-Dawley rats, aged 2 days, were randomly divided into three groups: sham-operation,WMI, and hUC-MSC ( n =18 each). Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in the white matter, and immunofluorescence staining was used to measure the expression level of ionized calcium-binding adapter molecule 1 (Iba1). Western blotting was used to measure the protein expression levels of inhibitory subunit of nuclear factor-kappa B alpha (IκBα), phosphorylated IκBα (p-IκBα), phosphorylated NF-κB p65 (p-NF-κB p65), myelin basic protein (MBP), and neuron-specific nuclear protein (NeuN). Quantitative real-time PCR was used to assess the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), MBP, and NeuN. Immunohistochemistry was used to measure the protein expression levels of MBP and NeuN. On day 28, the Morris water maze test was used to evaluate spatial cognitive ability., Results: Fourteen days after modeling, the sham-operation group exhibited intact white matter structure with normal cell morphology and orderly nerve fiber arrangement. In the WMI group, large-scale cell degeneration and necrosis were observed, and nerve fiber arrangement was disordered. The hUC-MSC group showed relatively normal cell morphology and more orderly nerve fibers. Compared with the sham-operation group, the WMI group had significantly higher proportions of Iba1-positive cells, increased protein levels of p-IκBα and p-NF-κB p65, and higher mRNA levels of TNF-α and IL-1β. The protein expression of IκBα and the positive expression of MBP and NeuN, as well as their protein and mRNA levels, were significantly reduced in the WMI group ( P <0.05). Compared with the WMI group, the hUC-MSC group showed reduced proportions of Iba1-positive cells, decreased protein levels of p-IκBα and p-NF-κB p65, and lower mRNA levels of TNF-α and IL-1β. Furthermore, IκBα protein expression and MBP and NeuN expression (both at the protein and mRNA levels) were significantly increased in the hUC-MSC group ( P <0.05). On day 28, the Morris water maze results showed that compared with the sham-operation group, the WMI group had significantly longer escape latency and fewer platform crossings ( P <0.05). In contrast, the hUC-MSC group had significantly shorter escape latency and more platform crossings than the WMI group ( P <0.05)., Conclusions: hUC-MSC transplantation can repair WMI in neonatal rats, promote the maturation of oligodendrocytes, and support neuronal survival, likely by inhibiting activation of the NF-κB signaling pathway mediated by microglial cells.

4-tert-Octylphenol (OP), an endocrine disrupting chemical is widely used in the production of industrial products. Prenatal exposure to endocrine-disrupting chemicals negatively affects the brain. However, the influence of OP exposure during neurodevelopment in adult offspring remains unclear. Thus, in the present study, we investigated the effects of maternal OP exposure on brain development in adult offspring by analyzing primary glial cell cultures and mice. Our findings revealed that OP exposure led to a specific increase in the mRNA expression of the ionized calcium-binding adapter molecule 1 (Iba-1) and the proportion of amoeboid microglia in the primary glial cell culture and adult offspring mice. Exposure to OP increased the transcriptional activation of Iba-1 and estrogen response element, which were counteracted by estrogen receptor antagonists ICI 182,780. Moreover, OP exposure increased the nuclear localization of the estrogen receptor. Remarkably, OP exposure decreased the mRNA expression levels of proinflammatory cytokines and genes associated with immune response in the brains of the offspring. OP exposure upregulated actin filament-related genes and altered cytoskeletal gene expression, as demonstrated by microarray analysis. The morphological changes in microglia did not result in an inflammatory response following lipopolysaccharide treatment. Taken together, the effects of OP exposure during neurodevelopment persist into adulthood, resulting in microglial dysfunction mediated by estrogen receptor signaling pathways in the brains of adult offspring mice., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eui-Man Jung reports financial support was provided by the National Research Foundation of Korea (NRF) funded by the Korean Government (MSIT) (grant number: 2021R1C1C100328611) and Global - Learning & Academic research institution for Master’s·PhD students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2023-00301938)., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tubular aggregate myopathy (TAM) is a rare myopathy characterized by muscle weakness and myalgia. Muscle fibers from TAM patients show characteristic accumulation of membrane tubules that contain proteins from the sarcoplasmic reticulum (SR). Gain-of-function mutations in STIM1 and ORAI1, the key proteins participating in the Store-Operated Ca 2+ Entry (SOCE) mechanism, were identified in patients with TAM. Recently, the CASQ1 gene was also found to be mutated in patients with TAM. CASQ1 is the main Ca 2+ buffer of the SR and a negative regulator of SOCE. Previous characterization of CASQ1 mutants in non-muscle cells revealed that they display altered Ca 2+ dependent polymerization, reduced Ca 2+ storage capacity and alteration in SOCE inhibition. We thus aimed to assess how mutations in CASQ1 affect calcium regulation in skeletal muscles, where CASQ1 is naturally expressed. We thus expressed CASQ1 mutants in muscle fibers from Casq1 knockout mice, which provide a valuable model for studying the Ca 2+ storage capacity of TAM-associated mutants. Moreover, since Casq1 knockout mice display a constitutively active SOCE, the effect of CASQ1 mutants on SOCE inhibition can be also properly examined in fibers from these mice. Analysis of intracellular Ca 2+ confirmed that CASQ1 mutants have impaired ability to store Ca 2+ and lose their ability to inhibit skeletal muscle SOCE; this is in agreement with the evidence that alterations in Ca 2+ entry due to mutations in either STIM1, ORAI1 or CASQ1 represents a hallmark of TAM., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R , agrB/D , and eutV/W . The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

This study aimed to investigate the expression and functional role of nesfatin-1, a peptide hormone traditionally associated with appetite regulation, in the human endometrium. Specifically, we examined its presence and regulatory potential in the human endometrial stromal cell line, THESC cells, focusing on the process of endometrial decidualization, which is critical for implantation and pregnancy maintenance. We found that nesfatin-1 and its binding sites were expressed in THESC cells. Furthermore, nesfatin-1 protein expression decreased after treatment with 17β-estradiol but increased upon exposure to progesterone, indicating an influence of sexsteroid hormones on nesfatin-1 expression. Notably, administration of nesfatin-1 protein to THESC cells resulted in significant upregulation of genes associated with decidualization, such as insulin-like growth factor binding protein 1 (IGFBP1) and prolactin. In addition, our research showed that nesfatin-1 promotes decidualization through the activation of the FAK/PI3K/AKT signaling pathway. These findings underscore the central role of nesfatin-1 in endometrial decidualization, and suggest its potential utility in the development of new treatments to improve fertility and pregnancy outcomes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hyunwon Yang reports administrative support, article publishing charges, equipment, drugs, or supplies, statistical analysis, travel, and writing assistance were provided by Seoul Women’s University. Hyunwon Yang reports a relationship with Seoul Women’s University that includes: employment. Hyunwon Yang has patent None pending to None. I declare that there is no conflict of interest regarding the publication of this manuscript. The research was conducted without any influence or support from external organizations or entities that could bias the results or interpretation of the findings. No financial, personal, or professional relationships that could be perceived as having influenced the research or conclusions have been involved. If there are other authors, they 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.)

The influence of extracellular matrix (ECM) stiffness on cell behavior is a well-established phenomenon. Tumor development is associated with the stiffening of the ECM. However, the understanding of the role of biomechanical behavior and mechanotransduction pathways in the oncogenesis of tumor cells remains limited. In this study, we constructed in vitro models using Polydimethylsiloxane substrates to create soft and stiff substrates. We then evaluated the migration of lung cancer cells A549 using video-microscopy and transwell assays. The mechanical properties were assessed through the utilization of atomic force microscopy, Optical Magnetic Twisting Cytometry, and traction force analysis. Additionally, the expression of Calponin 3 (CNN3) was evaluated using reverse transcription‑quantitative PCR and immunofluorescence techniques. Our observations indicate that the presence of a stiff substrate enhances A549 motility, as evidenced by increased stiffness and traction force in A549 cells on the stiff substrate. Furthermore, we observed a decrease in CNN3 expression in A549 cells on the stiff substrate. Notably, when CNN3 was overexpressed, it effectively inhibited the migration and invasion of A549 cells on the stiff substrate. The results of our study provide novel perspectives on the mechanisms underlying cancer cell migration in response to substrate mechanical properties., (© 2024 Wiley Periodicals LLC.)

Background: Vascularization after rib fracture is a crucial physiological process that is essential for the repair and healing of the rib. Studies have shown that CD90 plays a critical role in regulating rib fracture healing, but the underlying mechanism of its role has not been fully elucidated., Methods: CD90 adenovirus knockout mice were used to construct a rib injury model. The bone healing was observed by micro-CT. CD31/EMCN immunofluorescence staining was performed on bone tissue to observe the density of H-shaped and L-shaped blood vessels at the site of bone injury. CD31 and EMCN dual-stained single cells from the rib fracture sites were detected by flow cytometry. The periosteal stem cells transfected with CD90 or Notch1 overexpression and silencing vector were co-cultured with osteoblast MC3T3-E1 in osteogenic induction medium. Moreover, bone microvascular endothelial cells were extracted from the rib injury and co-cultured with the periosteal stem cells transfected with CD90. CCK-8 was used to detect cell viability, RT-qPCR and Western blot were used to detect Notch1, Notch2, Notch3, Notch4, CD31, HIF-1α, CD90, RUNX2, OCN and OPN expression. Alkaline phosphatase (ALP) staining and alizarin red staining were used to observe mineralized nodules. Immunofluorescence staining was used to detect the expression of Dll4, Notch, and CD90 in each group of cells. The angiogenesis experiment was conducted to observe cellular vascular formation., Results: Compared with the Adsh-NC group, the bone healing in the Adsh-CD90 group was significantly impaired, with a marked reduction in the number and volume of blood vessels at the rib fracture site, as evidenced by CD31/EMCN immunofluorescence staining, which showed a reduction in the number of H type vessels at the site of bone injury. It was found that CD90 depletion can inhibit the signaling of Dll4/Notch in the rib fracture site. Furthermore, we found that overexpression of Notch1 reverses the impairment of tubule formation in bone microvascular endothelial cells caused by CD90 suppression.r.Dll4 protein reverses the inhibitory effect of CD90 deletion on periosteal stem cells and MC3T3-E1 cell viability and osteogenesis. In the end, we found that overexpression of Notch1 and CD90 can promote angiogenesis of bone microvascular endothelial cells and Notch pathway activation., Conclusion: CD90 can affect vascular formation in mouse rib fractures, and CD90 may be regulated by Dll4/Notch., Competing Interests: Declarations. Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Institutional review board: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All the animal works were approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (XHEC-NSFC-2022-418). Informed consent: Not applicable., (© 2024. The Author(s).)

Lysosomal damage induces stress granule (SG) formation. However, the importance of SGs in determining cell fate and the precise mechanisms that mediate SG formation in response to lysosomal damage remain unclear. Here, we describe a novel calcium-dependent pathway controlling SG formation, which promotes cell survival during lysosomal damage. Mechanistically, the calcium-activated protein ALIX transduces lysosomal damage signals to SG formation by controlling eIF2α phosphorylation after sensing calcium leakage. ALIX enhances eIF2α phosphorylation by promoting the association between PKR and its activator PACT, with galectin-3 inhibiting this interaction; these regulatory events occur on damaged lysosomes. We further find that SG formation plays a crucial role in promoting cell survival upon lysosomal damage caused by factors such as SARS-CoV-2 ORF3a , adenovirus, malarial pigment, proteopathic tau, or environmental hazards. Collectively, these data provide insights into the mechanism of SG formation upon lysosomal damage and implicate it in diseases associated with damaged lysosomes and SGs., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Calcium- and integrin-binding protein 1 (CIB1) has a diverse role in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. It is associated with cancer, cardiovascular disease and male infertility. Here, CRISPR/Cas9 genome-editing technology was employed to establish a CIB1 knockout human embryonic stem cell line, which exhibited normal pluripotency and karyotype., 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 B.V. All rights reserved.)

Background: Fibulin-2 (FBLN2) is a secreted extracellular matrix (ECM) glycoprotein and has been identified in the mouse mammary gland, in cap cells of terminal end buds (TEBs) during puberty, and around myoepithelial cells during early pregnancy. It is required for basement membrane (BM) integrity in mammary epithelium, and its loss has been associated with human breast cancer invasion. Herein, we attempted to confirm the relevance of FBLN2 to myoepithelial phenotype in mammary epithelium and to assess its expression in molecular subtypes of human breast cancer., Methods: The relationship between FBLN2 expression and epithelial markers was investigated in pubertal mouse mammary glands and the EpH4 mouse mammary epithelial cell line using immunohistochemistry, immunocytochemistry, and immunoblotting. Human breast cancer mRNA data from the METABRIC and TCGA datasets from Bioportal were analyzed to assess the association of Fbln2 expression with epithelial markers, and with molecular subtypes. Survival curves were generated using data from the METABRIC dataset and the KM databases., Results: FBLN2 knockdown in mouse mammary epithelial cells was associated with a reduction in KRT14 and an increase in KRT18. Further, TGFβ3 treatment resulted in the upregulation of FBLN2 in vitro. Meta-analyses of human breast cancer datasets from Bioportal showed a higher expression of Fbln2 mRNA in claudin-low, LumA, and normal-like breast cancers compared to LumB, Her2 +, and Basal-like subgroups. Fbln2 mRNA levels were positively associated with mesenchymal markers, myoepithelial markers, and markers of epithelial-mesenchymal transition. Higher expression of Fbln2 mRNA was associated with better prognosis in less advanced breast cancer and this pattern was reversed in more advanced lesions., Conclusion: With further validation, these observations may offer a molecular prognostic tool for human breast cancer for more personalized therapeutic approaches., (© 2024. The Author(s).)

Corneal dystrophies are phenotypically and genetically heterogeneous, often resulting in visual impairment caused by corneal opacification. We investigated the genetic cause of an autosomal dominant corneal stromal dystrophy in a pedigree with eight affected individuals in three generations. Affected individuals had diffuse central stromal opacity, with reduced visual acuity in older family members. Histopathology of affected cornea tissue removed during surgery revealed mild stromal textural alterations with alcianophilic deposits. Whole genome sequence data were generated for four affected individuals. No rare variants (MAF < 0.001) were identified in established corneal dystrophy genes. However, a novel heterozygous missense variant in exon 4 of SPARCL1, NM&#95;004684: c.334G > A; p.(Glu112Lys), which is predicted to be damaging, segregated with disease. SPARC-like protein 1 (SPARCL1) is a secreted matricellular protein involved in cell migration, cell adhesion, tissue repair, and remodelling. Interestingly, SPARCL1 has been shown to regulate decorin. Heterozygous variants in DCN, encoding decorin, cause autosomal dominant congenital stromal corneal dystrophy, suggesting a common pathogenic pathway. Therefore, we performed immunohistochemistry to compare SPARCL1 and decorin localisation in corneal tissue from an affected family member and an unaffected control. Strikingly, the level of decorin was significantly decreased in the corneal stroma of the affected tissue, and SPARCL1 appeared to be retained in the epithelium. In summary, we describe a novel autosomal dominant corneal stromal dystrophy associated with a missense variant in SPARCL1, extending the phenotypic and genetic heterogeneity of inherited corneal disease., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: This study was approved by the Research Ethics Committees of University College London (UCL) (22/EE/0090) and Moorfields Eye Hospital (MEH) London (13/LO/1084) and was conducted in accordance/compliance with the Declaration of Helsinki. A written informed consent form was obtained from all participants who provided peripheral blood or corneal tissue samples. Control corneal tissue was similarly isolated from donor corneoscleral discs rejected for clinical transplantation (Miracles in Sight Eye Bank, TX, USA)., (© 2024. The Author(s).)

Mutimo cylindricus gametes have two flagella with different structures : an anterior and a posterior flagellum. Their flagellar waveforms are regulated by calcium ions through various mechanisms, however the factors involved in this regulation remain largely unknown To elucidate the molecular basis underlying the difference between the two flagella, we performed a flagellar proteomic analysis of male M. cylindricus gametes. We identified 848 proteins shared with Ectocarpus siliculosus, including 28 calcium-binding proteins. Among the EF-hand proteins, a 111 kDa protein showed predominant localization along the anterior flagellum. Immunogold localization suggested that this protein is associated with outer doublet microtubules. This is the first report to show heterogeneous localization of a calcium-binding protein between two flagellar axonemes and suggests that calcium-binding proteins are involved in the specific regulation of the anterior flagellum., 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 GmbH. All rights reserved.)

Calcific deposits in the arterial media have been associated with a number of metabolic and genetic disorders including diabetes, chronic kidney disease and generalized arterial calcification of infancy. The loss of matrix Gla protein (MGP) leads to medial elastic lamina calcification (elastocalcinosis) in both humans and animal models. While MGP-deficient (Mgp -/- ) mice have been used as a reliable model to study medial elastocalcinosis, these mice are difficult to maintain because of their fragility. Also, these mice are unsuitable for long-term calcification studies in relation to age and sex as most often they die prematurely. In order to circumvent these problems we generated Mgp -/- ;ApoE-FGF23 mice, which in addition to the ablation of Mgp alleles, carries a transgene expressing the phosphaturic hormone FGF23. Increased FGF23 levels in the circulation and ensuing hypophosphatemia in these mice lead to a complete prevention of medial calcification until late adulthood. Interestingly, upon feeding a high phosphorus diet for 10 days, we were able to induce medial calcification in 3-week-old Mgp -/- ;ApoE-FGF23 mice. Our mineral analyses showed that the Ca/P% in the calcific deposits in these mice were comparable to that of 5-week-old Mgp -/- mice although the level of crystallinity differed. The aorta explants from Mgp -/- ;ApoE-FGF23 mice resulted in elastocalcinosis in the presence of 2 mM phosphate in the culture medium which was completely prevented by pyrophosphate analogue alendronate. Mgp -/- ;ApoE-FGF23 mice will be suitable for future in vivo or ex vivo studies examining the effects of age, sex and mineralization inhibitors on medial elastocalcinosis., 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 Inc. All rights reserved.)

Introduction: Preeclampsia (PE) is a pregnancy complication marked by high blood pressure, posing risk to maternal and fetal health. "Genomic imprinting", an epigenetic phenomenon regulated by DNA methylation at Differently Methylated Regions (DMR's), influences placental development. Research on circulating extracellular vesicles (EVs) in PE suggests them as potential source for early biomarkers, but methylation status of EV-DNA in Preeclampsia is not reported yet., Methods: This study examines the methylation and expression profile of imprinted genes - PEG10, PEG3, MEST, and DLK1 in circulating EVs of 1 st and 3 rd trimester control and early onset preeclampsia (EOPE) pregnant women (n = 15) using pyrosequencing and qRT-PCR respectively., Results: In 1 st trimester, PEG3 was significantly hypermethylated, whereas no significant methylation changes were noted in PEG10 and MEST in EOPE. In 3 rd trimester, significant hypomethylation in PEG10, PEG3 and IGDMR was observed whereas significant hypermethyaltion noted in MEST. mRNA expression of PEG10, PEG3 and DLK1 was not affected in circulating EVs of 1 st trimester EOPE. However, in 3 rd trimester significant increased expression in PEG10, PEG3 and DLK1 noted. MEST expression was reduced in 3 rd trimester EOPE. No correlation was observed between average DNA methylation and gene expression in PEG10 and PEG3 in 1 st trimester. However, in 3 rd trimester, significant negative correlation was noted in PEG10 (r = -0.426, p = 0.04), PEG3 (r = -0.496, p = 0.01), MEST (r = -0.398, p = 0.03) and DLK1 (r = -0.403, p = 0.03)., Discussion: The results of our study strengthen the potential of circulating EVs from maternal serum as non-invasive indicators of placental pathophysiology, including preeclampsia., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)