The thyroid axis orchestrates key biological functions in fish, including metamorphosis. Disruption of thyroid signaling by endocrine disrupting compounds (EDCs), particularly estrogens, remains understudied during this sensitive period. This study compared the thyroidal effects of two estrogens: 17α-ethinylestradiol (EE2), a synthetic compound widely used in combined oral contraceptives (COCs), and estetrol (E4), a natural estrogen produced only during pregnancy and recently introduced as the estrogenic component of a new COC. Zebrafish (Danio rerio) were exposed to EE2 and E4 at concentrations ranging from 10 to 10,000x their respective measured (0.1 ng/L for EE2) or predicted (32 ng/L for E4) environmental levels from fertilization to 30 days post-fertilization (dpf). Samples were collected at 5 dpf for proteomic analysis to assess effects on thyroid organogenesis and early function, and at 14, 22, and 30 dpf to evaluate growth, thyroid histology, hormone levels, and transcriptomic profiles, thereby examining the effects on thyroid function throughout metamorphosis. Proteomic analysis at 5 dpf showed no disruption of thyroid organogenesis or function following exposure to either estrogen. However, both compounds induced concentration-dependent differentially expressed proteins (DEPs) linked to key developmental and metabolic pathways, with possible long-term effects on metamorphosis. DEPs were mainly associated with Rab signaling (RAB23, RAB35B, RAB38B), retinoic acid metabolism (CRABP1B, RDH5, RDH10A/B), mTOR signaling (RHEB, IGF1RB, BCL2A), oxidative stress (GPX9, TXNRD2.2, DAO.1/2, GNPAT), and energy metabolism (COX15, COX4I2). During metamorphosis, EE2 (≥ 100 ng/L) significantly reduced larval growth and thyroid signaling activity, as shown by modulation of thyroid-axis gene expression. In contrast, E4 did not affect growth or thyroid structure up to 320,000 ng/L, and triggered only modest transcriptomic changes in thyroid-axis genes at 32,000 ng/L. This study represents the first comparative assessment of EE2 and E4 on fish thyroid using a multiparametric approach at early and later developmental stages. These findings demonstrate that while both compounds influenced early developmental and metabolic pathways, only EE2 disrupted thyroid signaling during metamorphosis and induced phenotypic impairments. E4 caused weaker effects on the thyroid axis and did not induce any observable metamorphic disruptions. Overall, E4 appears to pose a lower environmental risk despite its early proteomic impact.

Glycoprotein hormones alpha 2 (GPA2) and beta 5 (GPB5) are considered to be ancestral members of the glycoprotein hormone (GPH) family. Despite their potential roles in regulating thyroxine production, the immune response, and ovarian function, their roles in the testis remains largely unknown. To further explore this regulation, catshark testicular explants containing late spermatid stages were treated in vitro with recombinant GPA2, GPB5 and single-chain GPB5#GPA2. The comparative proteomic analysis of the treated explants revealed that ScGPB5 and ScGPB5#ScGPA2 were the most effective, modulating abundance of 449 and 525 proteins, respectively. In contrast, ScGPA2 only modulated 212 proteins. Among the differentially abundant proteins (DAPs), 103 showed substantial abundance changes in response to at least one treatment, with mean Log2ratios of 2.5 and - 2.5. Manual functional annotation linked these proteins to spermiogenesis and immunity. Key proteins and candidates involved in spermiogenesis were highlighted, including SPINK2, which is involved in acrosome protection, and LRRC69, which is related to human spermatogenic failure. These results reinforce the hypothesis of a GPA2/GPB5 paracrine regulation of catshark spermiogenesis. They also underscore the necessity of functional and comparative studies to elucidate the physiological functions of GPA2/GPB5 across a broad spectrum of taxa, from non-vertebrates to mammals. SIGNIFICANCE: To our knowledge, this study is the first to report the effects of stimulation with recombinant ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2 on vertebrate testicular tissue in vitro. A large-scale, untargeted proteomic analysis revealed for the first time that all three-ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2-can modulate the abundance of 103 of the 7619 identified proteins, including proteins related to spermiogenesis and immunity, as well as others that are less well characterized. This report reinforces the importance of functionally characterizing GPA2 and GPB5 by showing the first results toward the characterization of their spermatogenesis related function in a specie of evolutive interest, the small-spotted catshark. In the future, a complete characterization of GPA2 and GPB5 will further our understanding of the functional evolution of glycoprotein hormones-a keystone group of hormones for vertebrate physiology and critical targets for fertility modulation in both livestock and humans.

Chondrosarcoma (CHS), the second most common primary malignant cartilage tumor, is largely resistant to conventional therapies, making surgical resection the standard treatment. Proton therapy offers a physical advantage through the Bragg peak, enabling targeted irradiation while sparing surrounding tissues. However, differential biological responses between malignant and normal cartilage cells remain poorly understood. In this study, CHS SW1353 cells and normal chondrocytes (MC615) were exposed to proton irradiation. Biological responses were assessed via clonogenic survival, cell viability, apoptosis (caspase 3/7), micronucleus formation, cell cycle profiling, and oxidative stress markers. Proteomic changes were analyzed using mass spectrometry and bioinformatics. CHS cells exhibited higher radioresistance (D10 = 6.45 Gy) than normal chondrocytes (D10 = 5.08 Gy), oxidative stress adaptation, G1 arrest and proteomic plasticity, whereas normal chondrocytes displayed increased oxidative stress, extracellular matrix fragility and impaired integrin signaling. Notably, the tumor-specific increased levels of Tyrosine-protein kinase Fyn and Yes1-associated transcriptional regulator (YAP1) signaling suggest molecular drivers of radioresistance. Overall, proton irradiation elicits distinct biological and proteomic responses in malignant versus normal cartilage cells. These findings highlight potential radiosensitization targets, including Fyn/Src and YAP1/Hippo pathways, while underscoring the need to optimize proton therapy to enhance tumor control while minimizing damage to healthy cartilage.

Anthropogenic underwater noise is a growing environmental stressor in coastal ecosystems, yet its molecular effects on invertebrate early life stages remain poorly understood. Using a data-independent acquisition proteomic workflow, we characterized the proteome of blue mussel (Mytilus edulis) post-larvae and examined changes in protein abundance following exposure to realistic cargo-shipping noise. A total of 7249 proteins were identified, of which 902 showed significant abundance differences across low, medium, and high sound pressure levels (i.e., 121, 127, and 151 dB re 1 μPa, respectively). Functional enrichment and interaction analyses revealed coordinated, intensity-dependent changes in proteins involved in metabolic, cytoskeletal, and regulatory processes. Forty-nine proteins were consistently regulated across treatments, including candidates associated with developmental regulation, morphogenesis, and shell-related pathways, indicating a conserved molecular response to acoustic exposure. Those results provide a reference proteomic dataset for M. edulis post-larvae and highlight proteome-level plasticity associated with shipping-related acoustic disturbance during metamorphosis.

Calciprotein particles (CPPs) are calcium- and phosphate-containing nanoparticles numbers of which are increased in patients with chronic kidney disease (CKD). CPPs have been associated with the development of vascular disease, although the underlying mechanisms are unknown. We previously showed that CPPs induce endothelial cell (EC) dysfunction by reducing nitric oxide (NO) bioavailability and generating superoxide (O2 .-). Here, we tested the hypothesis that CPPs induce mitochondrial calcium (Ca2+) overload, which may trigger mitochondrial dysfunction and, consequently, EC activation. Exposure of human umbilical vein ECs to CPPs resulted in significantly increased cytosolic and mitochondrial Ca2+ levels compared to vehicle-treated ECs. Proteome analysis demonstrated impaired endoplasmic reticulum calcium signalling, and decreased enrichment of proteins in the mitochondrial OXPHOS complexes I-III in CPP-exposed ECs. Respirometry data confirmed these findings and demonstrated decreased basal and maximal respiration in CPP-exposed ECs. This was accompanied by reduced mitochondrial membrane potential, reduced antioxidant capacity and loss of mitochondria. In the presence of cyclosporin A, a potent mitochondrial permeability transition pore inhibitor, CPP-induced EC activation and cell death were attenuated. Taken together, our data indicate that CPP-induced Ca2+ overload is an important trigger of mitochondrial dysfunction, and EC activation and cell loss, which eventually may contribute to the development of vascular diseases in CKD. Interventions that target CPP-induced mitochondrial dysfunction might preserve EC function and possibly alleviate the development of vascular diseases in CKD. KEY POINTS: Calciprotein particles (CPPs) are calcium- and phosphate-containing nanoparticles numbers of which are increased in patients with chronic kidney disease and which have been associated with the development of vascular disease. In this study, we tested the hypothesis that CPPs induce mitochondrial calcium (Ca2+) overload in endothelial cells, thereby triggering mitochondrial dysfunction and endothelial activation. We show that exposure of HUVECs (human umbilical vein endothelial cells) to CPPs results in increased cytosolic and mitochondrial Ca2+ levels, which is associated with alterations in mitochondrial processes (proteome analysis), cellular respiration, mitochondrial integrity and number. CPP-induced EC activation and cell death were attenuated in the presence of cyclosporin A, a potent mitochondrial permeability transition pore inhibitor. Our data indicate that CPP-induced Ca2+ overload triggers mitochondrial dysfunction, endothelial activation and cell loss. Interventions that target CPP-induced mitochondrial dysfunction might preserve EC function in chronic kidney disease.

In the context of current global change, variations in water temperature are one of the environmental conditions with serious consequences for marine life, including reproductive processes. In the small spotted catshark Scyliorhinus canicula, spermatogenesis occurs in spermatocysts composed of synchronously developing germ cells associated with Sertoli cells, forming a zonal arrangement of the spermatogenic wave. Male catsharks are known to show little to no seasonal variation in spermatogenesis, unlike other sharks that may have a seasonal break at the spermatogonia-primary spermatocyte transition, creating a zone of degeneration (ZD). Unexpectedly, an unusually high number of male catsharks, collected in the eastern English Channel in September 2022, exhibited a ZD. Analysis of bottom water temperatures indicated that the warmest period, from July to September 2022, was higher than the average for previous years. To further explore the biological perturbations associated with the ZD of the testis, a histological description and a comparative proteomic analysis of the ZD with a zone of intact mitosis-meiosis transition were performed. The results showed that only the spermatogonia at stages II to IV were degenerating, as well as their associated Sertoli cells, while the rest of the testicular tissue appeared not impacted. This spermatogonia-specific degeneration was also supported by the proteomic analysis, which showed that only 20% (1565 proteins) of the identified proteins presented a change in abundance. Based on functional annotations, the comparative proteomic analysis revealed cell cycle disruption, impaired DNA damage repair, apoptosis and stimulated lipid metabolism. In addition, follicle-stimulating hormone and luteinizing hormone receptors were upregulated in the ZD. In conclusion, the results showed a correlation between high water temperatures and an arrest of spermatogenesis in S. canicula, with implications for the conservation of elasmobranchs.

N6-adenosine RNA methylation (m6A) is a key regulator of gene expression during embryogenesis and neurogenesis in mammals and insects. However, its functional relevance remains unknown in lophotrochozoans like the Pacific oyster Crassostrea gigas, despite its association with developmental gene expression. We treated oyster embryos with STM2457, a METTL3 methyltransferase-inhibitor. m6A-RNA reduction in treated embryos induced morphological alterations and 5-HT immunohistochemistry revealed impaired neuronal development. Transcriptome and proteome analyses indicated that m6A inhibition disturbs transcription and translation. Epitranscriptome sequencing showed that m6A inhibition increased transcript length by exon and intron retention, suggesting m6A-dependent recruitment of splicing factors at intron-exon boundaries. Together, our results support an essential role for m6A in neural differentiation and development in the oyster by regulating alternative splicing. This study provides the first evidence of a functional role for m6A in lophotrochozoan development, providing new eco-evo-devo insights of epitranscriptomic processes.

Chondrosarcomas (CSs) are resistant to conventional chemotherapy and radiotherapy. Therefore, new therapeutic approaches are needed. The aim of this study was to validate the use of adenosine analogs as a new therapeutic strategy for the treatment of CS. Five adenosine analogs (aristeromycin, cladribine, clofarabine, formycin, and pentostatin) were evaluated in vitro on CS cell lines via both (two-dimensional) 2D cultures and three-dimensional (3D) alginate bead models. Cell viability was assessed by cell counting or ATP assays. Apoptosis was measured and cell cycle analyzed. The most promising compounds were further tested in vivo using a xenograft CS model in nude mice. Four analogs significantly reduced the viability of CSs. Among these, cladribine and clofarabine demonstrated potent efficacy in both 2D and 3D models by inducing apoptosis. Cladribine was further found to induce cell-cycle arrest, leading to apoptosis-mediated cell death. In vivo, both cladribine and clofarabine exhibited substantial antitumor effects in a xenograft model. In conclusion, cladribine and clofarabine, which have already been approved for clinical use in leukemia and multiple sclerosis, are promising candidates for the treatment of CS. Their efficacy in preclinical models suggests that these molecules could be repurposed for phase 2 clinical trials in patients with CS.

Autophagy is essential for homeostasis and nutrient recycling. Its activity increases with aging and in response to deficiencies. The effects of defective autophagy on root metabolism have not yet been described. Addressing this question through root proteome analyses, we found that most V-ATPases were less abundant in the roots of autophagy mutants than in wild type. V-ATPases deficit, associated with lower root water contents and lower nitrate, magnesium, and potassium concentrations, indicated that the disturbance of cellular ion and water management in autophagy mutants was likely related to vacuole function. Isotopic δ13C analyses and leaf temperature measurements using thermography showed that water deficit in autophagy mutants was not due to excess transpiration, as the conductance of stomata was reduced in mutants compared to wild type. Many proteins related to the catabolism of amino acids and lipids and the tricarboxylic acid (TCA) cycle were over-abundant in atg mutants. The increase in several proteases that paralleled amino acid catabolism suggested that in the absence of autophagic flux, compensatory processes could be established to degrade proteins, recycle amino acids, and fuel TCA. Whether the V-ATPases defect affects energy metabolism and promotes lipid and amino acid catabolism to compensate and fuel TCA remains to be explored. In conclusion, this report establishes for the first time a correlation between autophagy and vacuole function through V-ATPases, particularly with regard to water and ion management. Additionally, this report shows the exacerbation of amino acid catabolism in relation to the stimulation of the TCA cycle in autophagy mutants.

BACKGROUND:The adaptation of the redox system and bioenergetics is a major factor contributing to cancer metabolism. Redox therapy is promising but still requires molecular studies that consider the reactive species interactome (RSI) concept, which integrates reactive oxygen, nitrogen, sulfur, carbonyl species, and redox enzymes. Our aim was to decipher the role of the RSI in glioblastoma (GBM), including by challenging the RSI with the MnTBAP redox agent. METHODS:The effects of MnTBAP on the redox system and bioenergetics were investigated on several GBM models, namely in vitro 2D culture, in vitro 3D culture with two human GBM tumoroids, and in vivo preclinical model, which included male and female comparisons. RESULTS:We show - for the first time - that MnTBAP represses the sulfide:quinone oxidoreductase (SQOR) involved in the sulfur metabolism and bioenergetics, and targets the RSI through the sulfido-redox system. Through in vitro silencing and overexpression approaches, we also demonstrate that SQOR contributed to GBM cell growth and that its decrease is involved in the molecular effect of MnTBAP. Consequently, MnTBAP induces a switch between apoptosis, uncontrolled necrosis, and ferroptosis depending on the glioblastoma models. CONCLUSION:Our findings represent the next step in establishing a better understanding of redox biology in the context of GBM.

Microplastics (MPs) are increasingly associated with physiological disruptions in aquatic organisms, yet the biological responses to environmentally sourced particles remain underexplored. This study investigated the reproductive toxicity of environmentally derived MPs collected from the Ikopa River (Antananarivo, Madagascar) in Danio rerio. Zebrafish were chronically exposed to cryomilled riverine MPs (1.2-50 mu m) at concentrations of 100 and 1000 mu g/L for 66 days, with daily reproductive assessments conducted over the final 21 days in accordance with OECD Test Guideline 229. Microplastic accumulation in gonadal tissue was assessed, along with subcellular responses via enzymatic assays in gonads and proteomic profiling in liver samples. Reproductive toxicity was evaluated through gonadal histology, fecundity, and fertility rates. MPs accumulated in gonads in a sex-and concentration dependent manner, with the highest levels in males exposed to 1000 mu g/L (177.88 +/- 102.65 particles/mg tissue, mean +/- SD, n = 4). Despite MPs accumulation, no histopathological lesions were observed. However, significant oxidative stress and energy metabolism disruptions were identified in the liver, suggesting hepatic dysfunction as a potential driver of reproductive impairments. Furthermore, six polychlorinated biphenyl (PCB) congeners ranging from dozens to hundreds of ng/g MPs, and seven polybrominated diphenyl ether (PBDE) congeners in the range of a few ng/g MPs were detected on MPs surfaces, which may exacerbate toxicity via apoptosis inhibition. These findings provide novel mechanistic insights into how environmentally relevant MPs impair reproductive function in fish. The results underscore the necessity of incorporating environmental microplastics into toxicity testing frameworks to ensure accurate ecological risk assessment.

The pituitary glycoprotein hormones (GPHs) control several physiological processes in vertebrates such as reproduction and metabolism. They include the luteinizing hormone (LH), the follicle-stimulating hormone (FSH), and the thyroid-stimulating hormone (TSH), which activate their cognate leucine-rich repeat G protein-coupled receptors (LGRs), LHR, FSHR, and TSHR. Each GPH consists of a common α subunit and a specific βFSH, βLH or βTSH subunit. More recently, two supplementary GPH proteins, GPA and GPB, were identified in nearly all bilaterians and are the ancestors of the pituitary GPH α- and β-subunits, respectively. Chondrichthyans (holocephalans and elasmobranchs), the sister group of bony vertebrates, are the most ancient clade to possess diversified GPH subunits. In the present study, GPA2, GPB5, TSHβ2, but not TSHβ1, and TSHR sequences have been identified in several elasmobranch genomes, and their 3D models were analyzed. Functional hormone-receptor interactions were studied in the small-spotted catshark (Scyliorhinus canicula) and showed that conditioned media from cells expressing the recombinant single-chain ScGPB5-ScGPA2 were more effective than independent subunits in activating ScTSHR, ScFSHR, and ScLHR. Expression profiles were analyzed by real-time PCR, in situ hybridization, and immunohistochemistry along the male genital tract, other male and female tissues, and female tissues. A broader tissue distribution expression was observed for tshr and gpa2 than for gpb5, which was mainly observed in the testes. In testis, expression of tshr and gpb5 by Sertoli cells and of gpa2 by germ cells suggested paracrine/autocrine functions of GPA2/GPB5/GPHR signaling during spermatogenesis. This study complements the data on GPA2 and GPB5 by studying a chondrichthyan of phylogenetic interest for understanding the evolution of endocrine regulation in vertebrates.

Despite the considerable decline of cervical cancer incidence in developed countries, the disease remains a public health problem in low-income and middle-income countries due to the low popularity of human papillomavirus vaccination and cervical cancer screening. Mainly treated with radiotherapy, the number of recurrences linked to radioresistance increases in women suffering from this disease and constitutes major obstacle. Here, we perform a combined proteomic and phosphoproteomic profiling of HeLa cervical cancer cells after in vitro treatment with X-rays and carbon ions. We observed differential and extensive alterations at the proteins and phosphoproteins levels. In total, we observed 96 and 102 differentially expressed proteins (DEPs) after X-rays and C-ions irradiation, respectively. For phosphoproteins, our results revealed 21 and 41 DEPs in response to C-ions and X-rays ionizing radiation respectively. Furthermore, our study revealed several mechanisms significantly activated by cells in response to ionizing radiation, potentially related to cancer radioresistance, including sister chromatid segregation, rRNA processing, ribosomal large subunit biogenesis, positive regulation of phagocytosis, engulfment, peptidase regulatory activity and negative regulation of ERK1/2 cascade. We also identified three proteins IPM3, DUSP3 and COQ7, oppositely expressed across the C-ions and X-rays groups while MX2 phosphorylation was downregulated in both radiation qualities. Finally, our study revealed a specific kinase signature, associated with Hela cells radioresistance: CDK5, MTOR and CDK2 kinases were predicted in the group of X-rays irradiation while CDK1, PLK1 SRC and MAPK1 kinases were predicted in the group of C-ions irradiation. Taken together, these findings could help to define new potential pathways and biomarkers to be targeted in the treatment of cervical cancer. Insight Box Statement of Integration, Innovation and Insight In this study, a robust proteomic and phospho-proteomic strategy was developed in order to display HELA cells responses to radiations. Two time points were selected to highlight the early responses of cells, following irradiation with low and high LET. CDK1, SRC, MAPK1 kinases were predicted to be activated in response to carbon ions irradiation, while CDK5, MTOR, ATM kinases were predicted in response to X-rays. Several accessions, playing pivotal role in cell proliferation and resistance, were upregulated in X-rays irradiated cells and down regulated in carbon ions irradiated cells. This study gives an accurate picture of molecular events linked with HELA cells radioresistance and offer potential drug targets for optimization of cervical cancer radiotherapy.

Hypothalamic gonadotropin-releasing hormone (GnRH) regulates the production of gonadotropins, which control reproduction. In elasmobranchs, unlike other gnathostomes, GnRH is released into the systemic circulation to stimulate gonadotrope cells located in the ventral lobe of the pituitary. The aim of this study was to investigate the potential role of systemic GnRH in the regulation of the testis in Scyliorhinus canicula. Phylogeny and synteny analyses identified three GnRHs and four GnRH receptor (ScGnRHR-I1, -IIa1, -IIa2 and -IIb2). In vitro functional hormone-receptor interactions using synthetic ScGnRHs showed that all ScGnRHs were effective at receptors, except ScGnRHRIIa2, at femtomolar to nanomolar concentrations, with lower efficiency for ScGnRH1/ScGnRHRIIb2. Real-time PCR analyses in a wide range of tissues, including male and female reproductive tracts, showed that all three gnrh were expressed mainly in the brain and all four gnrhr were expressed in the testis, particularly during spermiogenesis. Testicular explants containing cysts with spermatids were treated with ScGnRHs and their protein content analyzed by NanoLC-ESI-MS/MS, highlighting 1677 significantly differentially expressed proteins. Among them, the growth hormone receptor (GHR) and proteins involved in cholesterol and steroid metabolism, including several HSD17bs, were upregulated. In situ hybridization showed that ghr, hsd17b3 and hsd17b12 transcripts were localized in Sertoli cells, which are the main testicular steroidogenic cells in S. canicula. Fifteen steroids were assayed in the culture media, using LC-ESI-HRMS/MS, and an increase in 17β-estradiol concentrations was observed, consistent with hsd17b expressions. Furthermore, proteins involved in transcription and DNA structure were downregulated in response to GnRHs. In conclusion, this study showed that ScGnRHs may play a direct role in the regulation of elasmobranch testes by promoting spermiogenesis and modulating steroidogenesis.

Seeds of Brassicaceae produce a large diversity of beneficial and antinutritional specialized metabolites (SMs) that influence their quality and provide resistance to stresses. While SM distribution has been described in leaves and root tissues, limited information is available about their spatiotemporal accumulation in seeds. Camelina sativa (camelina) is an oilseed Brassicaceae cultivated for human and animal nutrition and for industrial uses. While we previously explored SM diversity and plasticity, no information is available about SM distribution and expression of related proteins and genes in camelina seeds. In this study, we used a multi-omic approach, integrating untargeted metabolomics, proteomics, and transcriptomics to investigate the synthesis, modification, and degradation of SMs accumulated in camelina seed tissues (seed coat, endosperm, embryo) at six developmental and two germination stages. Metabolomic results showed distinct patterns of SMs and their related pathways, highlighting significant contrasts in seed composition and spatial distribution for the defense-related and antinutritional glucosinolate (GSL) compounds among camelina, Arabidopsis thaliana, and Brassica napus, three closely related Brassicaceae species. Notably, thanks to metabolomic and proteomic/transcriptomic techniques the variation in GSL spatial distributions was primarily driven by differences in their structure (metabolomics data) and transport (transcriptomic and proteomic data) mechanisms. Long-chain C8-C11 methylsulfinylalkyl GSLs were predominantly accumulated in the seed coat and endosperm, while mid- and short-chain C3-C7 methylsulfinylalkyl GSLs were accumulated in the embryo. Characterizing the spatial dynamics of seed SMs provides valuable insights that can guide the development of crops with optimized distribution of beneficial and toxic metabolites, improving seed nutritional profiles.

Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, has gained attention as a promising therapeutic target in osteoarthritis (OA) due to its central role in modulating inflammation, catabolism, and hypertrophy within chondrocytes. Previous studies have further demonstrated that EZH2 inhibition can slow OA progression in surgically induced mouse models, highlighting its potential in reducing joint degradation. However, the precise mechanisms by which EZH2 influences other key cell types in OA pathology remain poorly understood. In this study, we aimed to evaluate the effects of EZH2 inhibition in an alternative OA model and investigate its broader impact on cellular and molecular pathways across various tissues involved in OA progression and joint pain. OA was induced in mice via intra-articular injection of monosodium iodoacetate (MIA), with disease progression evaluated by histological and behavioral assessments. In parallel, human synoviocytes and bone marrow-derived cells were isolated from OA patients. Synoviocytes were stimulated with interleukin-1β (IL-1β) in the presence or absence of the EZH2 inhibitor EPZ-6438 (Tazemetostat), and ChIP-Seq and proteomic analyses were conducted to identify genomic and proteomic targets of EZH2. Additionally, the effects of EZH2 inhibition on M1 macrophage polarization and osteoclast differentiation were analyzed. Results revealed that EZH2 inhibition attenuated both OA progression and joint pain in the MIA-induced mouse model. IL-1β stimulation significantly upregulated EZH2 expression in synoviocytes, and treatment with the EZH2 inhibitor reduced the expression of genes linked to inflammation, pain, and catabolism while promoting autophagy. Proteomic analysis highlighted significant alterations in pathways related to IL-1β signaling, matrix metalloproteinase (MMP) activation, and autophagy, as well as changes in proteins associated with metabolic regulation and axon guidance. Importantly, EZH2 inhibition decreased M1 macrophage polarization and osteoclast formation, cellular processes that contribute to OA pain and inflammation. In conclusion, this study underscores the pivotal role of the histone methyltransferase EZH2 in the pathophysiology of osteoarthritis and associated joint pain. Our findings reveal that EZH2 inhibition not only attenuates inflammation in synovial cells and macrophages but also modulates axon guidance and osteoclastogenesis, both critical in OA progression and pain. These insights position EZH2 inhibition as a promising, multi-targeted therapeutic approach for addressing the complex cellular interactions underlying osteoarthritis, offering new hope for effective treatment strategies in this debilitating condition. ### Competing Interest Statement The authors have declared no competing interest.

Seeds are crucial for plant reproduction, dispersal and agriculture. Seed quality and vigour greatly impact crop production by enabling rapid and uniform germination under various environmental conditions. This leads to healthy seedlings that can withstand both biotic and abiotic stresses, which are particularly important in the context of the accentuation of global climate change. Upon imbibition during germination sensu stricto , seeds release exudates, complex mixtures of organic and inorganic molecules, into the microenvironment surrounding them, known as the spermosphere. These exudates play a pivotal role in seedling development and overall plant fitness by influencing microbial selection, growth and interactions in the spermosphere, ultimately shaping the plant's microbiome. Proteins such as enzymes with protection properties have previously been demonstrated to be released by the seeds in their exudates. However, limited information is available pertaining to peptides in seed exudates. Here, we developed an experimental protocol to extract and identify peptides in the spermosphere of germinating common bean seeds. We showed that our methodology was successful in identifying a broad spectrum of peptides and that extraction solvent choice impacts peptide identification both qualitatively and quantitatively. We also show the possibility of using online prediction tools to predict the properties of identified peptides based on their amino acid sequence. We propose that this approach may be used to identify potential molecules that could be used as candidates for developing strategies to enhance seed quality and improve crop productivity.

While the ability of plastic particles to transport heavy metals is well established, their Trojan horse effect on aquatic organisms remains debated, as they are suspected of facilitating the penetration of chemicals in tissues but also of reducing bioavailability and accelerating pollutant elimination. Here, we investigated the combined effects of 250 nm polystyrene nanoplastics (NPs) and methylmercury (MeHg) on zebrafish larvae over a 30-day exposure period. Larvae were exposed to 1000 μg/L NPs, 1 μg/L MeHg (MeHg1), 10 μg/L MeHg (MeHg10), or their respective combinations (Mix1 and Mix10). The presence of NPs enhanced MeHg accumulation and redirected its distribution toward the fish's head and eyes. On their own, NPs altered swimming activity, while MeHg10 induced mortality, reduced growth and diminished swimming activity. Proteomic analysis highlighted significant effects on lipid metabolism, oxidative stress, detoxification, myogenesis and catabolism. Although no light sensitivity deficits were detected through visual motor response testing, proteomic data suggested vision impairment in the mixture-exposed groups. High mortality rates were observed in Mix10-exposed fish, likely due to severe hypoactivity, which hindered feeding. This hypoactivity was linked to disrupted lipid metabolism, impaired neurotransmission, reduced ATP production, and neuroinflammation leading to neuronal degeneration. We concluded that the presence of NPs intensified MeHg neurotoxicity over a prolonged exposure, significantly increasing mortality.

BACKGROUND:Vascular calcification is highly prevalent in Chronic Kidney Disease (CKD) and is associated with markedly increased cardiovascular risk. High serum phosphate in CKD increases calcification propensity via generation of circulating calciprotein particles (CPP2), crystalline nanoaggregates composed of calcium, phosphate, and serum proteins. CPP2 induce vascular calcification in vascular smooth muscle cells (VSMCs) in vitro. In vivo, endothelial cells, rather than VSMCs are primarily exposed to CPP2, yet understanding the influence of endothelial cells on vascular calcification is limited. METHODS:We investigated calcification-promoting signalling by endothelial cells on VSMCs. Effects of CPP2 exposure to endothelial cells on CPP2 uptake, endothelial cell activation, and endothelial cell-derived secretome were studied. Effects of the secretome on VSMC calcification were investigated. Using NanoString nCounter analysis the effects of CPP2-activated endothelial cell-conditioned medium on VSMCs gene expression were mapped. RESULTS:Endothelial cells internalise CPP2 and elevate ICAM-1, E-selectin, and VCAM-1-mRNA expression, indicating endothelial activation. VSMCs cultured in conditioned medium from CPP2-activated endothelial cells demonstrated enhanced calcification, suggesting that CPP2-activated endothelial cells release pro-calcifying soluble factors. Mass spectrometry was utilized to identify 1171 proteins in the CPP2-activated endothelial cells' secretome. Among these, 76 proteins were differentially expressed compared to control endothelial cells' secretome, including proteins related to blood vessel development, extracellular matrix remodelling, and oxidative stress-related processes. Finally, endothelial cell-derived paracrine factors present in conditioned medium enhanced mRNA-expression of calcification-related factors in VSMCs. CONCLUSIONS:CPP2-activated endothelial cells promote VSMC calcification via paracrine signalling. In response to these paracrine factors, VSMCs increase the expression of pro-calcification genes.

In Gnathostomes, reproduction is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis, with the involvement of the pituitary gonadotropic hormones (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which activate their cognate receptors, FSHR and LHR, expressed in gonads. Each GTH consists of a common α subunit and of a specific FSHβ or LHβ subunit. Chondrichthyes (holocephalans and elasmobranchs) is a sister group of bony vertebrates. This position is highly favorable for the understanding of the evolution of endocrine regulations of reproduction among gnathostomes. Surprisingly, the characterization of gonadotropins and their receptors is still limited in chondrichthyes. In the present study, GTH and GTHR sequences have been identified from several chondrichthyan genomes, and their primary structures were analyzed relative to human orthologs. 3D models of GTH/GTHR interaction were built, highlighting the importance of the receptor hinge region for ligand recognition. Functional hormone-receptor interactions have been studied in HEK cells using the small-spotted catshark (Scyliorhinus canicula) recombinant proteins and showed that LHR was specifically activated by LH whereas FSHR was activated by both FSH and LH. Expression profiles of GTHs and their receptors were explored by real-time PCR, in situ hybridization and immunohistochemistry during spermatogenesis, along the male genital tract and other tissues, as well as in some female tissues for comparison. Tissue-expression analyses showed that the highest levels were observed for fshr transcripts in testis and ovary and for lhr in specific extragonadal tissues. The two receptors were expressed at all stages of spermatogenesis by both germ cells and somatic cells, including undifferentiated spermatogonia, spermatocytes, spermatids, somatic precursors and Sertoli cells; differentiated Leydig cells being absent in the testis of S. canicula. Receptors were also expressed by the lymphomyeloid epigonal tissue and the testicular tubules. These results, suggest a wide range of gonadotropin-regulated functions in Elasmobranchs, as well as functional redundancy during spermatogenesis. These extended functions are discussed in an evolutionary context in which the specificity of gonadotropin signaling must have contributed to the evolution of gonadal cells' morphology and function.