Background: Self-harm, which includes both non-suicidal self-injury and suicidal behaviors, poses a major global public health challenge. This study provides a comprehensive analysis of trends in self-harm worldwide, the socioeconomic disparities associated with it, and future projections, using data from the Global Burden of Disease, Injuries, and Risk Factors Study (GBD) 2021. Methods: Self-harm data were extracted from GBD 2021, including incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life years (DALYs) for 204 countries and territories from 1990 to 2021. Age-standardized rates and estimated annual percentage change (EAPC) were calculated. Inequality was assessed using the Slope Index of Inequality (SII) and Concentration Index (CI). Autoregressive Integrated Moving Average (ARIMA) models were employed to generate projections of self-harm burden from 2022 to 2040. Results: The global burden of self-harm is projected to change substantially by 2040, with deaths estimated to increase to 829,853 (95% Uncertainty Interval (UI), 262,233-1,397,474) and prevalence projected to rise to 35,863,341 (95% UI, 8,079,108-63,647,574) cases (representing a 131.9% increase from the 2021 baseline of 15,467,153 cases). From 1990 to 2021, age-standardized rates of self-harm demonstrated decreasing trends globally and across sociodemographic index (SDI) levels, with the largest declines observed in high-middle SDI countries. Gender disparities were evident, with more pronounced decreases in females. Inequalities in DALYs due to self-harm decreased over time but remained higher among females in lower-SDI populations. Conclusions: Despite decreasing age-standardized rates, the global burden of self-harm is projected to increase substantially by 2040, with driven by increasing incidence and prevalence in incidence and prevalence. Inequities persist, particularly among females in lower-SDI populations. Implementation of targeted prevention and intervention strategies, strengthening of mental health systems, and addressing social determinants of health are imperative to reduce the growing burden of self-harm worldwide.

Uveal melanoma (UM), a rare yet aggressive ocular malignancy in adults, highlights the critical need for targeted therapies to improve clinical outcomes. Elevated FGFR1 expression in UM correlates with aggressive disease progression and poor survival outcomes, underscoring its therapeutic value. This study reports the development of [68Ga]Ga-DOTA-cHW8, an FGFR1-specific PET tracer derived from ligand-based computational design of cyclic peptide cHW8, enabling noninvasive quantification of FGFR1 expression in UM xenograft models. Bioinformatics analysis of clinical databases and immunoblotting of clinical specimens confirmed FGFR1 as an important biomarker of UM. The radiotracer [68Ga]Ga-DOTA-cHW8 demonstrated superior pharmacokinetics with rapid tumor accumulation, high binding specificity, and rapid renal clearance. In vitro specificity assays demonstrated targeted accumulation of [68Ga]Ga-DOTA-cHW8 in FGFR1-positive 92.1 cells and UM PDC cells. In vivo microPET imaging validated tumor-specific accumulation of [68Ga]Ga-DOTA-cHW8 in FGFR1-positive UM xenografts (including PDX models), which was blocked by an unlabeled peptide. Furthermore, [68Ga]Ga-DOTA-cHW8 validated FGFR1 dynamic modulation during erdafitinib treatment in CDX and PDX models, establishing its efficacy for noninvasive UM treatment response assessment. This study reports a cyclic peptide-based radiotracer, [68Ga]Ga-DOTA-cHW8, for FGFR1 PET imaging in UM. Through rational design and preclinical validation in UM models, we establish its high specificity, favorable pharmacokinetics properties, and capability to monitor FGFR1 dynamics during targeted therapy.

Approximately 50% of uveal melanoma (UM) patients develop treatment-resistant liver metastases, surviving less than one year after diagnosis. While BRCA1-associated protein 1 (BAP1) deficiency strongly correlates with UM metastasis, its mechanistic role remains unclear. Through integrated analysis of four UM cohorts and functional experiments validation, we identified S100 calcium binding protein A6 (S100A6) as a key metastasis-associated gene consistently upregulated in BAP1-deficient UM. Mechanistically, BAP1 deficiency enhances H2AK119ub deposition and Pol II recruitment at the S100A6 promoter, activating its transcription. Notably, we discovered that S100A6 functions as a novel ligand of fibroblast growth factor receptor 3 (FGFR3), triggering sustained signaling distinct from canonical ligands and activating inflammatory cancer-associated fibroblasts. Genetic or pharmacological targeting of S100A6-FGFR3 signaling effectively suppressed BAP1-deficient UM metastasis in preclinical models, highlighting the therapeutic potential of targeting this signaling pathway. Overall, our findings establish S100A6 as a critical mediator of hepatic metastasis in BAP1-deficient UM through FGFR3-dependent tumor microenvironment activation, revealing its therapeutic potential.

Background Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with limited therapeutic options. Circular RNAs (circRNAs) have emerged as critical regulators of cancer progression; however, the functional role of hsa_circ_0003472 in PDAC remains unexplored. Methods Expression of hsa_circ_0003472 was assessed in 30 paired PDAC and adjacent normal tissues and pancreatic cancer cell lines using quantitative RT-PCR. Loss-of-function experiments were performed to evaluate effects on proliferation (CCK-8, EdU), apoptosis (TUNEL, Western blot), migration, and invasion (Transwell assays). Gemcitabine sensitivity was determined by IC50 analysis. Bioinformatic prediction and dual-luciferase reporter assays identified the downstream regulatory axis. Xenograft mouse models validated findings in vivo. Results hsa_circ_0003472 was significantly upregulated in PDAC tissues and cell lines. Silencing hsa_circ_0003472 inhibited proliferation, migration, and invasion while promoting apoptosis and enhancing gemcitabine sensitivity. Mechanistically, hsa_circ_0003472 functioned as a competing endogenous RNA by sponging miR-1253, thereby relieving suppression of excision repair cross-complementing group 1 (ERCC1). Rescue experiments confirmed that the oncogenic effects of hsa_circ_0003472 were mediated through the miR-1253/ERCC1 axis. In vivo, hsa_circ_0003472 knockdown significantly reduced tumor growth and recapitulated molecular changes observed in vitro. Conclusion hsa_circ_0003472 promotes PDAC progression and chemoresistance through the miR-1253/ERCC1 regulatory axis, representing a potential therapeutic target for this devastating malignancy.

INTRODUCTION:Significant progress has been made in uncovering the role of DNA methylation in the development and ripening of fruit. However, whether DNA methylation is involved in litchi fruit ripening and in the corresponding mechanisms remain unknown. OBJECTIVES:The goal of this study was to explore the role of DNA methylation in the ripening, senescence, and the regulatory mechanism of litchi as well as to provide new perspectives for controlling litchi pericarp browning after harvest. METHODS:Here, single-base resolution maps of the DNA methylome during litchi fruit ripening and senescence was generated using whole genome bisulfite sequencing (WGBS). Association analysis of WGBS and RNA-sequencing was performed to reveal the potential regulatory effects of different regional methylation sites on gene expression in litchi in different fruit ripening and postharvest stages. A DNA methylation inhibitor, 5-azacytidine, treatment was also conducted. Meanwhile, the LcDML1 silencing mutant (TRV-LcDML1) was obtained using virus-induced gene silencing to confirm the regulatory role of LcDML1 in litchi ripening. RESULTS:Results showed that about 55%, 34%, and 11% of CG, CHG and CHH sites were methylated, respectively. Genome-wide DNA methylation increased during litchi fruit ripening and exhibited a regulatory role in gene expression during ripening but had limited influence during senescence. Furthermore, WGBS and RNA-sequencing analysis indicated that LcDML1 contributed to changes in genome-wide DNA methylation during litchi maturation. A 5-azacytidine treatment inhibited anthocyanin synthesis in litchi pericarp. In addition, LcDML1 silencing induced early coloration of litchi pericarp and upregulation of anthocyanin synthesis-related genes but downregulation of potential negative regulator of anthocyanin synthesis genes (NRAs). McrBC-PCR and dual-luciferase reporter assay suggested methylation regulation of NRAs promoters regulated the maturation and coloration of litchi fruit. CONCLUSION:LcDML1 is an important factor modulating the change of genome-wide DNA methylation as well as anthocyanin synthesis and participates in regulating litchi fruit ripening.

Uveal melanoma (UM) is the primary intraocular malignancy in adults and has an extremely poor prognosis due to a high rate of metastasis. Because current drug options are generally ineffective, there is an urgent need for new agents with anti-UM efficacy. The phenylcyclohexyl-urea UC2288 was investigated in in vitro, ex vivo and in vivo UM models. The anti-cancer actions of UC2288 were evaluated using cell viability and cell death assays. Tumour migration, invasion and reproductive cell growth assays were used to assess the anti-metastatic potential of UC2288. Such effects were corroborated in primary cultures derived from patient tumours and in vivo in a UM cell xenograft mouse model. UC2288 decreased UM cell proliferation in conventional and 3-dimensional cell culture by disrupting cell cycle progression and modulating cyclin expression. UC2288 also targeted the mitochondrion and increased the production of reactive oxygen species, which promoted necrotic cell death. In mechanistic studies, UC2288 activated AMPK and downstream eIF2/ATF pathways of ER stress and autophagy in UM cells. UC2288 also impaired UM cell migration, invasion and reproductive growth, which is consistent with anti-metastatic activity. These findings were replicated in vivo in a UM cell xenograft model. Taken together, UC2288 represents a promising candidate for further development that targets UM tumours with favourable anti-cancer effects.

Encapsulating the natural multi-pathway active compound pterostilbene (PT) within liposomes enhances its bioavailability. However, binding to lipids and the influence of temperature, crucial for food applications, remain poorly understood. Elucidating the interaction mechanism is essential for developing functional foods. Fluorescence spectroscopy and molecular dynamics simulation were employed to confirm the spontaneous binding of PT to lipids and identify its binding site within the hydrophobic tail region of the lipid bilayer. PT-loaded soybean lecithin liposomes (PT-LPs) were prepared using microfluidics, and heat treatment at 55 °C increased the encapsulation efficiency (EE%) from 57.88% to 66.18%. Furthermore, structural changes of PT-LPs during heating (20-90 °C) were investigated using 1-anilino-8-naphthalene sulfonate probe fluorescence and small-angle neutron scattering. Selecting an appropriate temperature for heat treatment improved PT EE%. This work advances our understanding of PT-liposome formation and stability, supporting the development of edible liposomal delivery systems exploiting the multi-pathway bioactivities of PT.

Rationale:Acute kidney injury (AKI) is a life-threatening clinical syndrome characterized by high mortality, in which tubular epithelial cell death represents a key pathological event. Emerging evidence underscores the importance of the ubiquitin system in the progression of AKI. Here, we focus on the function of YOD1 in AKI. Methods:We generated tubular epithelial cells (TECs)-specific Yod1 knockout mice (YOD1CKO) by crossing Yod1fl/fl mice and Ggt1-cre mice. Both YOD1CKO mice and Yod1fl/fl littermates were subjected to cisplatin- or ischemia/reperfusion (I/R)-induced AKI models. Through co-immunoprecipitation (Co-IP) combined with LC-MS/MS analysis, we identified potential substrate proteins of YOD1. Results:We observed that YOD1 is predominantly expressed in TECs and is upregulated during AKI injury. Renal tubular specific Yod1 knockout significantly alleviated tubular damage and apoptosis in AKI mice. Mechanistically, we identified the pro-apoptotic protein Bax as a direct substrate of YOD1. YOD1 removes K63-linked ubiquitin chains from Bax at lysine 128 via its catalytic cysteine residue C155, thereby promoting Bax activation, and mitochondrial translocation and subsequent apoptosis. YOD1 failed to promote apoptosis in Bax-deficient cells, confirming Bax as the essential downstream mediator. Conclusions:Our study reveals a previously unrecognized YOD1-Bax regulatory axis that drives tubular apoptosis in AKI, and highlights YOD1 may hold therapeutic potential.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder of the central nervous system (CNS) with a significant unmet need for new therapeutic options. Targeting embryonic ectoderm development (EED) is a potential novel therapeutic strategy for treating MS. Herein, based on optimization of potency and pharmacokinetic properties, we report a new class of trisubstituted pyridine derivatives as EED-H3K27me3 inhibitors for MS treatment. Notably, compounds 19 and 21 demonstrate potent EED binding affinity, significantly reduce H3K27me3 levels in dendritic cells (DCs), and effectively inhibit DC migration. Both compounds demonstrate appropriate pharmacokinetic (PK) properties, lymphoid tissue-targeting ability, and favorable in vivo safety profiles. Oral administrations of compounds 19 and 21 dose-dependently reduce spinal cord inflammation and alleviate disease progression in experimental autoimmune encephalomyelitis (EAE) mice. These results establish 19 and 21 as promising lead compounds for the development of EED inhibitors for MS therapy.

Colorectal cancer (CRC) ranks as the third most common malignancy worldwide, with metastasis representing the primary cause of mortality. Aberrant activation of the Wnt/β-catenin pathway drives epithelial‒mesenchymal transition (EMT) and CRC metastasis, making β-catenin a key therapeutic target. Josephin domain containing 2 (JOSD2), a deubiquitinase with protumorigenic roles in multiple cancers, has an undefined function in CRC metastasis. Herein, by screening a protein homeostasis-related inhibitor library, we identified HY041004, a reported JOSD2 inhibitor, which potently suppresses β-catenin transcriptional activity. Mechanistically, JOSD2 modulates β-catenin protein abundance and functional activity via the RAS-ERK signaling cascade rather than through direct deubiquitination of β-catenin. Functional assays further demonstrated that JOSD2 inhibition via RNA interference or pharmacological inhibition significantly attenuated CRC metastasis both in vitro and in vivo. Collectively, our findings identify JOSD2 as a critical oncogenic factor that promotes β-catenin activity and validate JOSD2 as an underlying therapeutic target for metastatic CRC (mCRC).

Sepsis-induced acute lung injury (ALI) involves complex pathological mechanisms. 5-methylcytosine (m5C) RNA modification, catalyzed by methyltransferases like NOP2, plays a crucial role in regulating inflammation and cellular processes. However, the role of NOP2 and its potential regulation of m5C modification in sepsis-induced ALI remains unclear. An in vitro ALI model was established by treating human pulmonary epithelial A549 cells with lipopolysaccharide (LPS). Inflammatory cytokine levels (IL-1β, IL-6, TNF-α) were measured by ELISA. Apoptosis was assessed by flow cytometry. Mitophagy was evaluated via immunofluorescence staining for mitochondrial Parkin and western blot analysis of Parkin, LC3-II, COX IV, and p62. The m5C modification of PINK1 mRNA was analyzed by m5C-RIP-PCR. The specific m5C site was identified using bioinformatics and validated by dual-luciferase reporter assays. LPS treatment significantly upregulated NOP2 expression in A549 cells. Knockdown of NOP2 attenuated LPS-induced inflammation, apoptosis, and promoted mitophagy, as evidenced by increased Parkin translocation, elevated LC3-II levels, and decreased p62 and COX IV levels. Mechanistically, NOP2 knockdown reduced m5C modification on PINK1 mRNA, particularly at site 197, thereby enhancing PINK1 mRNA stability and increasing its expression. Furthermore, knockdown of PINK1 reversed the protective effects of NOP2 knockdown on inflammation, apoptosis, and mitophagy in LPS-treated A549 cells. NOP2 is upregulated in LPS-induced ALI models. Its knockdown alleviates cellular injury by reducing the m5C modification of PINK1 mRNA, which enhances PINK1 expression and promotes mitophagy. The NOP2/m5C/PINK1 axis represents a novel regulatory pathway in sepsis-induced ALI, suggesting potential therapeutic targets for its treatment.

Natural cadinene-type sesquiterpenoids (CTSs) exhibit diverse structures and a wide range of pharmacological activities, including anti-tumor, anti-inflammatory, and antibacterial effects. They represent an important source for new drug discovery and development. By searching the PubMed, Web of Science, and the China National Knowledge Infrastructure (CNKI) databases, this review summarizes the research progress on 283 naturally occurring CTSs isolated and identified from plants, terrestrial fungi, marine organisms, and bacteria between 2015 and 2025. Based on their structural characteristics, these CTSs were classified into 145 bicyclic CTSs, 76 tricyclic CTSs, 37 carbon-bridged CTS dimers, 13 oxygen-bridged CTS dimers, 2 CTS trimers, and 10 heterocyclic CTSs. In addition, the biological activities and biosynthetic pathways of selected compounds are summarized. This work may facilitate the discovery and development of therapeutic agents derived from natural CTSs.

Colorectal liver metastasis (CRLM) remains the primary cause of mortality in patients with colorectal cancer (CRC), yet effective predictive tools and reliable biomarkers are still lacking. DeepMetabio-mCRC Screener, an integrated multi-omics framework combining large-scale transcriptomic profiles with serum metabolomics, was developed to address this gap. In a cohort of 1,077 CRC samples, 620 metabolism-related genes were used to train a convolutional neural network, yielding an area under the receiver operating characteristic curve of 0.92 in the validation cohort and 0.97 in the independent testing cohort, outperforming the performance of the 10 established machine learning models. Model-derived transcriptomic risk scores revealed 22 core metabolic features associated with metastatic progression and CRLM occurrence, particularly retinol and tryptophan metabolism. Cross-omics integration revealed aminocarboxymuconate-semialdehyde decarboxylase (ACMSD) as a promising biomarker associated with impaired nicotinamide adenine dinucleotide biosynthesis. Clinical validation in 100 CRC patients confirmed elevated ACMSD levels in patients with CRLM, which correlated with advanced stage, recurrence risk, an immune-inflamed tumor microenvironment, and heightened sensitivity to epidermal growth factor receptor/vascular endothelial growth factor receptor-targeted therapies. In vitro, ACMSD knockdown was associated not only with suppressed CRC cell migration caused by inhibition of the transforming growth factor-β/epithelial-to-mesenchymal transition pathway but also with decreased proinflammatory and immune-responsive pathways and reduced immune cell infiltration. These findings collectively validate the DeepMetabio-mCRC Screener as a substantial early risk prediction tool and underscore ACMSD, identified through this framework, as a multifunctional biomarker for diagnosis, prognosis, molecular characterization, and therapeutic decision-making in patients with CRLM.

Telomeres, coated by the shelterin complex, prevent end-to-end fusions and aberrant DNA repair, yet how telomere-binding proteins coordinate chromatin remodeling during the DNA damage response remains unclear. Here we show that the telomeric protein TRF1 is phosphorylated at serine 11 (S11) in response to DNA double-strand breaks, a modification that enhances cellular resistance to DNA damage. We found that CDK2 directly mediates this phosphorylation, which triggers recruitment of the histone methyltransferase SETD5 to telomeric chromatin. SETD5-dependent deposition of H3 trimethylation at lysine 36 (H3K36me3) promotes local chromatin decompaction and enables subsequent recruitment of the phosphatase PPP4C to dephosphorylate γH2AX. Loss of TRF1 S11 phosphorylation results in persistent γH2AX foci, delayed DNA repair, and compromised telomere integrity. Our results define a CDK2-TRF1-SETD5-PPP4C signaling axis that orchestrates phosphorylation-dependent chromatin remodeling at telomeres to ensure genome maintenance during DNA damage stress.

Background Ovarian cancer remains a leading cause of gynecologic cancer-related mortality, primarily driven by the development of multidrug resistance. Plant-derived natural products have gained attention as promising adjuncts to conventional treatments due to their multifaceted mechanisms and low systemic toxicity. Purpose This review summarizes recent advances in plant-derived compounds that counteract ovarian cancer resistance and explores their underlying molecular mechanisms. Methods A comprehensive literature search was performed in the PubMed and Web of Science databases to identify English-language articles published up to September 2025. The search terms included “ovarian cancer”, “drug resistance”, “aging”, “plant nanotechnology”, “Chinese herbal medicine”, and “natural products”, which were combined using Boolean operators “AND/OR”. Studies were eligible for inclusion if they addressed the pharmacological effects, molecular mechanisms, and therapeutic potential of plant-derived compounds in overcoming drug resistance in ovarian cancer. Basic research studies, in vitro and in vivo experiments, preclinical investigations, and relevant reviews were included, whereas duplicate articles were excluded. Results Various resistance mechanisms, including altered drug transport, enhanced DNA repair, immunosuppressive microenvironment remodeling, and senescence-associated phenotypes, are modulated by flavonoids, alkaloids, saponins, terpenes, and polysaccharides. These compounds target critical signaling pathways, such as PI3K/Akt/mTOR, STAT3, NF-κB, and VEGFR2/FAK. Advances in nanoparticle delivery and senescence regulation further enhance their therapeutic efficacy. Conclusions The integration of natural products with standard therapies may offer a viable strategy to overcome chemoresistance in ovarian cancer, highlighting the need for further translational and clinical investigations.

Traditional occupancy-driven pharmacology and emerging event-driven targeted protein degradation (TPD) play distinct roles in drug discovery. Although small-molecule inhibitors typically depend on sustained engagement of functional binding and often require systemic exposure, TPD induces selective protein elimination and can access targets that are refractory to conventional inhibition, namely, “undruggable” proteins. Expanding degradation mechanisms mediated by the ubiquitin‒proteasome system (UPS) and alternative pathways is therefore a central strategy for next-generation therapeutics. In this review, we provide an overview of the developmental trajectory of the TPD field and discuss how diverse modalities can be leveraged to address intracellular, membrane-associated, and extracellular protein targets. We summarize the relevant chemical design principles and translational challenges, with a focus on mitigating off-target toxicity, improving selectivity, and increasing bioavailability. The current evidence suggests that proteolysis-targeting chimeras, molecular glues, autophagy-targeting chimeras, and transferrin receptor-targeting chimeras could be key approaches. In addition, we discuss some innovative discovery platforms for expanding E3 ligase repertoires that may enable more rational degrader design. Finally, we highlight the current landscape of clinical trials and discuss opportunities and remaining hurdles for advancing TPD toward clinical translation.

KRAS is the most common mutated oncogenes in colorectal cancer (CRC), yet effective therapeutic strategies for targeting multiple KRAS mutations remained challenging. The prolonged protein stability of KRAS mutants contribute to their robust tumor-promoting effects, but the underlying mechanism is elusive. Herein by screening deubiquitinases (DUBs) siRNA library, we identify Josephin domain containing 2 (JOSD2) functions as a potent DUB that regulates the protein stability of KRAS mutants. Mechanistically, JOSD2 directly interacts with and stabilizes KRAS variants across different mutants, by reverting their proteolytic ubiquitination; while KRAS mutants reciprocally inhibit the catalytic activity of CHIP, a bona fide E3 ubiquitin ligase for JOSD2, thus forming a JOSD2/KRAS positive feedback circuit that significantly accelerates KRAS-mutant CRC growth. Inhibition of JOSD2 by RNA interference or its pharmacological inhibitor promotes the polyubiquitination and proteasomal degradation of KRAS mutants, and preferentially impede the growth of KRAS-mutant CRC including patient-derived cells/xenografts/organoids (PDCs/PDXs/PDOs) over that harboring wild-type KRAS. Collectively, this study not only reveals the crucial roles of JOSD2/KRAS positive feedback circuit in KRAS-mutant CRC, but also provides a rationale to target JOSD2 as the promising pan-KRAS-mutation-targeting strategy for the treatment of a broad population of CRC patients with KRAS variant across different mutant types.

Deubiquitinases (DUBs) catalyze the removal of ubiquitin moieties from substrate proteins, playing pivotal roles in regulating protein homeostasis. Targeting DUBs with small-molecule inhibitors to induce substrate protein degradation has emerged as a compelling strategy for addressing traditionally "undruggable" targets. Notably, multiple DUB inhibitors have advanced to preclinical and clinical stages. In our preliminary research, we discovered that the deubiquitinating enzyme Josephin domain-containing 2 (JOSD2) played a significant role in the development and progression of colorectal cancer (CRC). In this study, we identified a novel hit compound 1 targeting the JOSD2 through high-throughput screening of an internal compound library. Subsequently, guided by structure-activity relationship (SAR) analysis, compound 31 was synthesized, a JOSD2 inhibitor featuring a cyanamide warhead that selectively engaged the catalytic cysteine residue. Preliminary biological mechanism studies revealed a covalent binding mode between compound 31 and JOSD2. Further mechanistic studies have shown that 31 could induce the downregulation of KRAS protein expression in HCT116 cells, thereby exerting proliferation inhibitory activity (IC50 = 0.93 ± 0.01 μM). Collectively, our research identified that targeting JOSD2 with small-molecule inhibitors could represent a potential future therapeutic strategy for CRC.

Ethylene regulates fruit ripening at transcriptional, posttranscriptional, translational and posttranslational levels. However, the multiple regulatory mechanisms remain unclear. Here, we revealed a module that regulates fruit ripening transcriptionally and posttranslationally. Ethylene and abscisic acid markedly induced both transcript and protein levels of MaERF113 and MaABI5-like, and MaERF113 activated the expression of genes related to starch (MaGWD1, MaBAM3 and MaAMY3) and Chl (MaSGR1 and MaPPH) degradation. Their function and regulatory network in fruit softening were investigated using physiological, molecular biology and genetic approaches. Ectopic and transient overexpression of MaERF113 promoted fruit ripening and induced starch and Chl degradation in both banana and tomato, while silencing of MaERF113 caused an opposite effect. MaERF113 interacted with an E3 ubiquitin ligase MaSINAT5, which ubiquitinated MaERF113 at the K78 site, mediated its degradation and attenuated MaERF113-mediated transactivation of target genes. MaSINAT5 overexpression delayed fruit ripening, whereas MaSINAT5-silencing accelerated it. Interestingly, MaABI5-like activated and interacted with MaERF113 to enhance the promoter activity of genes involved in starch and Chl degradation. Overall, our findings uncovered a dynamic regulatory module of MaSINAT5/MaABI5-like-MaERF113 mediating 'Fenjiao' banana ripening by regulating starch and Chl degradation, which advanced our understanding of site-specific ubiquitination modification and phytohormone crosstalk in regulating fruit ripening.

Introduction: Banana fruit (Musa spp.) tend to suffer chilling injury but obvious differences exist in the cold tolerance among different banana genotypes. Cu-miRNAs refer to a group of miRNAs that target and regulate the expression of genes encoding copper-containing proteins, most of which play an important role in mediating cellular redox homeostasis. So far, whether Cu-miRNAs influence the differences in cold tolerance among fruit of different banana varieties remains unclear. Objectives: This work aimed to investigate the distribution and accumulation of Cu-miRNAs in the fruit of representative banana varieties with different genomic backgrounds, and their potential contribution to cold tolerance. Methods: AAA and ABB banana fruit were used to compare their cold tolerance phenotype and relevant physiological and biochemical properties. The distribution, structure, sequence conservation, and promoter characteristics of Cu-miRNAs were analyzed via bioinformatics. Next, the difference in Cu-miRNAs expression between ABB and AAA banana fruit by stem-loop qPCR was determined, and dual-luciferase reporter (DLR) experiment was performed to explore the upstream regulators of Cu-miRNAs. Finally, the function of miR528 in cold stress was verified by transient overexpression and silencing experiments on AAA and ABB banana peel slices. Results: ABB banana fruit contained higher levels of proline and glutathione (GSH). These antioxidants contributed to enhanced cold tolerance compared to AAA banana fruit. A higher abundance of most Cu-miRNAs, especially miR528, was observed in ABB than AAA banana fruit. Moreover, the positive regulators of these Cu-miRNAs, MaSPL4 and MaSPL5, were expressed at higher levels in ABB banana fruit under cold stress. Finally, transient transformation assays revealed that miR528 could alter the cold tolerance of both AAA and ABB banana fruit, serving as a positive regulator. Conclusion: Our findings confirm that Cu-miRNAs are closely related to the cold tolerance of banana fruit, offering potential target molecules for improving fruit cold stress resistance.