Search Results (56)

Lupinus angustifolius is an important ornamental plant; however, its poor tolerance to alkaline soils limits its cultivation and production. Based on the alkaline-tolerance-related Gshdz4-GsNAC019-GsEXPA8 regulatory module previously screened and identified in soybean, we used Agrobacterium rhizogenes-mediated transformation to overexpress in lupine roots the combinations Gshdz4-GsNAC019-GsEXPA8 (HNE), Gshdz4-GsNAC019 (HN), and GsNAC019-GsEXPA8 (NE) to investigate their effects on root development and alkaline tolerance. RT-PCR confirmed the successful generation of all overexpression lines. Under 100 mM NaHCO₃ stress, all overexpression lines exhibited less wilting and longer survival than the wild type (WT), with the HNE line showing the best phenotype. Physiological measurements showed that the overexpression lines had significantly higher proline content, antioxidant enzyme (SOD, CAT, POD) activities, and root activity, as well as lower malondialdehyde content. DAB and NBT staining of leaves indicated reduced accumulation of O₂⁻ and H₂O₂, suggesting enhanced antioxidant capacity. Root architecture analysis revealed that root length, surface area, volume, tip number, and fork number were significantly increased in HNE, HN, and NE lines compared with WT, with the most pronounced effect observed in HNE. Bioinformatics analysis and qPCR confirmed that Gshdz4 binds to and activates the promoter of the endogenous LaNAC072 (the lupine homolog of GsNAC019), while GsNAC019 binds to and activates the promoter of the endogenous LaEXPA8 (the lupine homolog of GsEXPA8), thereby triggering the endogenous alkaline tolerance regulatory mechanism. Furthermore, the overexpression combinations significantly upregulated the expression of alkaline stress-responsive genes, including LaSOS1, LaNHX6, LaP5CS, LaMYB39, and LaDnaJ1. This study provides theoretical support for molecular breeding of alkaline-tolerant lupine. Full article

Osteosarcoma (OS) is the most common primary bone cancer in adolescents and young adults. Despite tremendous preclinical and clinical efforts to advance therapy for OS, the standard of care, consisting of surgical resection and pre- and postoperative chemotherapy, has remained unchanged for over 40 years. Growing molecular understanding of OS highlights tumor heterogeneity as a major obstacle to therapeutic advances. In this narrative review, we comprehensively discuss current evidence of OS heterogeneity and strategies to overcome the barrier. Evidence shows that OS heterogeneity is multifactorial: it retains complex and dynamic somatic genomics, including genomic instability, alterations in tumor suppressors, and amplification/overexpression of oncogenes such as MYC. The tumor is associated with various germline vulnerabilities. OS’s tumor microenvironment has intense cellular and spatial diversity, which significantly shapes its heterogeneity. The effects of lineage plasticity, as well as epigenetic and metabolomic mechanisms, on OS heterogeneity are under study. To overcome this extreme heterogeneity, the therapeutic strategies for OS must be comprehensive and diversified. While surgical resection remains a mainstay of treatment, efforts to identify actionable biomarkers that guide risk stratification and therapy are ongoing. Diverse preclinical models offer insights into OS biology and novel therapeutics. To enhance combinational therapy for OS, various agents, including multi-targeted receptor tyrosine kinase inhibitors, immunotherapies, and epigenetic and metabolic modifiers, are being investigated. Distinctive efforts are continuing to establish maintenance therapy for OS. In summary, elucidating the complex drivers of OS heterogeneity, together with the development of multifaceted strategies to address them, is critical to accelerating therapeutic progress in OS. Full article

Fine particulate matter (PM_2.5) can directly impact pulmonary epithelial cells, resulting in lung injury. While it is known that PM_2.5 can alter the expression profile of microRNAs in the lung, its specific role in damaging pulmonary epithelial cells remains unclear. This study, therefore, employed RT-qPCR, Western blotting, and dual luciferase reporter assays to investigate the regulatory role of microRNAs in PM_2.5-induced cellular damage. PM_2.5 exposure induces oxidative stress, autophagy, and ferroptosis in rat lung alveolar epithelial cells (RLE-6TN). Further functional rescue experiments confirm that the ferroptosis-specific inhibitor Fer-1 can block PM_2.5-induced ferroptosis. Bioinformatics analysis and validation indicate that miR-212-5p plays a crucial role by negatively regulating RASSF1 through targeted inhibition. Overexpression of miR-212-5p activates the PI3K/AKT signaling pathway, thereby promoting autophagy and ferroptosis. However, when the expression of both miR-212-5p and RASSF1 is suppressed, PM_2.5-induced autophagy and ferroptosis are significantly alleviated by inhibiting the PI3K/AKT/mTOR signaling pathway. Rescue validation experiments demonstrated that, under PM_2.5 exposure combined with RASSF1 overexpression, miR-212-5p exacerbates the aforementioned cellular damage process. This study reveals that miR-212-5p regulates autophagy and ferroptosis by targeting RASSF1. These findings provide a multi-target intervention strategy for PM_2.5-related lung diseases. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by the absence of estrogen and progesterone receptors, as well as the lack of human epidermal growth factor 2 receptor overexpression. TNBC is associated with early onset, high metastatic potential, therapeutic resistance, and poor clinical outcomes exacerbated by the limited availability of effective targeted therapies. Advances in multi-omics profiling have further stratified TNBC into distinct molecular subtypes, each exhibiting unique genomic, epigenomic, and immune-related features that influence therapeutic responsiveness. This review explores the interplay between TNBC molecular heterogeneity, immune evasion mechanisms, and epigenetic regulation. TNBC demonstrates variable immunogenicity, with tumor-infiltrating lymphocytes serving as important prognostic and predictive biomarkers. However, immune escape commonly occurs through tumor microenvironment remodeling, T-cell exhaustion, cancer stem cell enrichment, and immune checkpoint pathways activation. Although immune checkpoint inhibitors have improved outcomes in selected patients, particularly in combination with chemotherapy, primary and acquired therapeutic resistance remain a significant challenge. Emerging evidence highlights the central role of epigenetic mechanisms in regulating immune-related gene expression and shaping the tumor immune microenvironment. Epigenetic silencing of antigen presentation machinery, interferon signaling pathways, and chemokine expression contributes to immune evasion and immunotherapy resistance. Importantly, pharmacological modulation of epigenetic regulators can restore immune recognition and induce “viral mimicry” through reactivation of endogenous retroelements, thereby enhancing antitumor immunity. Collectively, this review underscores the therapeutic potential of integrating epigenetic therapies with immunotherapy and chemotherapy to overcome immune resistance in TNBC. A deeper understanding of epigenetic-immune interactions may facilitate the development of more precise and effective treatment strategies tailored to TNBC molecular subtypes. Full article

Walnut is valued for being rich in nutrients and polyphenols, which are key bioactive metabolites; however, a comprehensive and dynamic assessment of metabolites in the husk and pellicle is still lacking. In this study, multi-omics approaches combining untargeted metabolomics and transcriptome analysis were conducted to systematically characterize the differential metabolite profile and regulatory networks in walnut husk and pellicle. Metabolomic profiling revealed a clear divergence in polyphenol compositions between the husk and the pellicle; the husk was predominantly enriched in nine phenolic acid compounds, whereas the pellicle accumulated eleven flavonoid compounds. Through co-expression network analysis, a transcription factor, JrMYB8, was identified and shown to act as a specific inhibitor and regulator of polyphenol biosynthesis. Functional characterization demonstrated that JrMYB8 overexpression significantly reduced the accumulation of the total phenol content (TPC) and the total flavonoid content (TFC) by directly repressing the expression of JrC4H. These findings not only provide a molecular target for manipulating polyphenol content in walnut tissues but also offer a target for improving flavor in walnut breeding. Full article

Background: DNMT3B is frequently overexpressed in molecular subsets of acute myeloid leukemia (AML) and is associated with poor prognosis. Unlike DNMT3A, DNMT3B is rarely mutated, suggesting dysregulation through epigenetic mechanisms. The regulatory basis and downstream consequences of DNMT3B overexpression in AML remain incompletely defined. Methods: We integrated analyses of BeatAML, TCGA, and BLUEPRINT cohorts with multi-omic profiling (RNA-seq, DNA methylation, ATAC-seq, and proteomics) in DNMT3B-high AML models. Nanaomycin A (NanA) was used as a DNMT3B-directed functional probe to interrogate cis-regulatory remodeling, transcriptional circuitry, and apoptotic dependencies. Results: DNMT3B overexpression was linked to enhancer-associated chromatin activation rather than recurrent genetic mutation, particularly in CEBPA- and NPM1-mutant AML. NanA exposure produced focal epigenomic remodeling, including 6900 differentially methylated CpGs, with 268 CpGs located within regions of altered chromatin accessibility. These changes were accompanied by coordinated transcriptomic and proteomic reprogramming enriched for cell-cycle, checkpoint, and stress-response pathways. Functionally, DNMT3B perturbation induced redistribution of cell-cycle phases with increased S-phase fraction and progressive apoptosis. Transcriptional profiling demonstrated induction of BH3-only sensitizers (NOXA, PUMA), repression of BCL2, and compensatory upregulation of MCL1 and BCL-XL, collectively reshaping apoptotic dependency. Combined DNMT3B perturbation and BCL2 inhibition produced cooperative cytotoxicity in DNMT3B-high AML models. Conclusion: DNMT3B functions as a context-dependent epigenetic regulator linking enhancer-associated chromatin organization with proliferative control and apoptotic resistance in AML. DNMT3B-directed epigenetic perturbation remodels cis-regulatory circuitry and is associated with increased venetoclax responsiveness, supporting DNMT3B-governed networks as a candidate co-targeting axis in high-risk AML. Full article

Background/Objectives: Melanoma is one of the deadliest types of skin cancer due to its ability to metastasize if not treated early. While targeted- and immune- therapies have significantly improved melanoma treatment outcomes, acquired drug resistance even with combined therapeutics remain prevalent. SIRT6 is a nuclear histone deacetylase that regulates DNA repair, metabolism, and chromatin remodeling. It is overexpressed in melanoma and its inhibition in melanoma is known to have anti-proliferative response, and alterations in pathways related to cell cycle, senescence, and metastasis. Methods: To deepen our understanding of the role of SIRT6 in melanoma, in this study we utilized RNA sequencing, proteomics, and Ingenuity Pathway Analysis on genetically modified human melanoma cells to determine the downstream mechanism of SIRT6 in melanoma. Results: SIRT6 knock down (KD) in A375 and G361 melanoma cells, with CRISPR/Cas9 or shRNA techniques, resulted in a significant decrease in proliferation and clonogenic survival of the cells. SIRT6 KD caused an altered expression of multiple genes associated with cell proliferation, mitotic regulation, invasion, cell death/senescence, and immunomodulation, including AURKB, ANLN, MYC, FOXM1, RABL6, E2F2, TP53, RBL1, OSM, TNF, IL1B, IL6, and IFNG. Comparative analysis at both transcription and translation levels revealed coordinated downregulation of proliferation, invasion, and migration and upregulation of targets related to cell death, apoptosis, and necrosis. Multi-omics analysis also predicted downregulation of signaling networks associated with MAP3K20, MYC, MKNK, and HMGCR. Conclusions: Given its involvement in tumorigenesis, this study underlines the importance of SIRT6 in melanoma and provides support to its potential as a novel therapeutic target for melanoma. Full article

Background/Objectives: Hepatocellular carcinoma (HCC) therapies are limited by poor response, rapid resistance, and recurrence of aggressive disease. Sorafenib, a multi-tyrosine kinase inhibitor, can trigger β-catenin stabilization and activation, contributing to resistance. Overexpression of the chemokine receptor CXCR6 and its ligand CXCL16 and hyperactivation are implicated in HCC progression and β-catenin stabilization. We hypothesized that SBI-457, a small-molecule CXCR6 antagonist we developed, could disrupt CXCR6/β-catenin crosstalk and enhance sorafenib sensitivity. Methods: We tested SBI-457 alone and in combination with sorafenib in SK-Hep-1 xenograft models and a panel of human HCC cell lines. Tumor burden, β-catenin activation, and CXCR6 expression were assessed by tumor volume measurements, immunohistochemistry, Western blotting, and immunofluorescence. Soluble CXCL16 levels were quantified by ELISA, and cell death responses were evaluated using MTT assays. Results: In vivo, SBI-457 combined with sorafenib reduced normalized tumor volume by 55% compared to vehicle controls, modestly exceeding monotherapy effects, and attenuated sorafenib-induced β-catenin upregulation. In vitro, SBI-457 blocked nuclear accumulation of β-catenin and reversed sorafenib-induced increases in β-catenin levels. Enhanced cell death was observed in specific “responder” HCC cell lines (Hep-3B, SNU-398, JHH-5), which correlated with high intracellular β-catenin, secretion of soluble CXCL16, and expression of a high molecular weight form of CXCR6. In contrast, “non-responder” cell lines with conventional CXCR6 expression and low CXCL16 secretion showed no enhanced cell death response. Conclusions: CXCR6 antagonism with SBI-457 can modulate β-catenin activation and may help overcome sorafenib resistance in selected HCC models. These findings support further development of CXCR6 antagonists as single agents or combination therapies to improve treatment outcomes in HCC. Full article

Background: Adhesion G protein-coupled receptor G6 (ADGRG6), also known as GPR126, has been implicated in several malignancies. However, its expression pattern, clinical significance, and mechanistic role in pancreatic adenocarcinoma (PAAD) remain unclear. Methods: We combined multi-omics analyses, tissue microarray immunohistochemistry, and a series of functional experiments, including 2D and 3D spheroid cultures, zebrafish xenografts, and murine tumor models—to investigate the expression, clinical significance, and mechanism of ADGRG6 in PAAD. The association between ADGRG6 expression and immune infiltration was assessed using TIMER and GEPIA databases, followed by mechanistic validation through ADGRG6 modulation in PAAD cell lines. Results: ADGRG6 was significantly overexpressed in PAAD and correlated with larger tumor size, higher grade, advanced TNM stage, and poor overall survival. Multivariate logistic regression confirmed that high ADGRG6 expression was independently associated with higher pathological grade. Functionally, ADGRG6 silencing markedly inhibited PAAD cell proliferation, migration, and invasion in both 2D and 3D cultures, as well as in zebrafish and nude mouse xenograft models. Integrated transcriptomic and immune analyses revealed that ADGRG6 expression positively correlated with mast cells, macrophages (M1/M2), Th2/Th17 subsets, and interferon–responsive neutrophils. Mechanistically, ADGRG6 silencing reduced STAT6 phosphorylation and GATA3 expression, consistent with the suppression of the NF-κB→STAT6→GATA3 axis. Conclusions: ADGRG6 functions as an oncogenic driver in PAAD, promoting tumor progression and fostering an immunosuppressive microenvironment via NF-κB/STAT6 signaling. These findings not only broaden the mechanistic understanding of ADGRG6 function but also suggest it as a promising target for therapeutic intervention in PAAD. Full article

To enhance the L-threonine synthesis level in Escherichia coli, this study constructed screening markers rich in L-threonine rare codons. By replacing all the threonine codons in the protein sequences with a high proportion of threonine with L-threonine rare codons and linking them to the fluorescent proteins with the same replacement, high-throughput screening of L-threonine production mutant strains was achieved. To address the metabolic imbalance caused by overexpression of a single enzyme, an artificial multi-enzyme complex system was constructed based on the principle of cellulosome self-assembly. By co-locating ThrC-DocA and ThrB-CohA, the substrate transfer path was shortened, achieving a 31.7% increase in L-threonine production. Furthermore, combined with multi-copy chromosomal integration technology via CRISPR-associated transposase (MUCICAT) technology, the thrC-docA-thrB-cohA gene cluster was integrated into the genome of the high-yield strains obtained through screening, eliminating the plasmid-dependent metabolic burden and significantly enhancing genetic stability. The modular assembly of metabolic pathways by using cellulosome elements provides a new paradigm for the optimization of complex pathways and lays a theoretical and technical foundation for the efficient production of L-threonine. Full article

RAC3 encodes a small Rho-family GTPase essential for cytoskeletal regulation and neurodevelopment, and de novo RAC3 variants typically act as gain-of-function alleles that cause severe neurodevelopmental disorders. In this study, we analyzed a fetus with multisystem congenital anomalies and identified a de novo RAC3 p.(T17R) variant by genome sequencing. To elucidate the pathogenicity of this variant, we combined in silico variant prioritization, structural and energetic modeling, and pathogenicity prediction with in vitro biochemical assays, including GDP/GTP exchange, GTP hydrolysis, effector pull-down, and luciferase reporter analyses in COS7 cells, as well as morphological analysis of primary hippocampal neurons. Furthermore, we performed in vivo analyses using a mouse in utero electroporation to assess cortical neuron migration, axon extension, and dendritic development. Our biochemical results suggest that RAC3-T17R exhibits markedly increased GDP/GTP exchange, with a preference for GDP binding, and undetectable GTP hydrolysis. The mutant displayed minimal binding to canonical RAC effectors (PAK1, MLK2, and N-WASP) and failed to activate SRF-, NFκB-, or AP1-dependent transcription. Neuronal overexpression of RAC3-T17R impaired axon formation in vitro, while in vivo expression delayed cortical neuron migration and axon extension and reduced dendritic arborization. Clinically, the fetus exhibited corpus callosum agenesis, microcephaly, organomegaly, and limb contractures. Collectively, these findings indicate that the RAC3 p.(T17R) variant may represent a signaling-deficient allele with pleiotropic, variant-specific mechanisms that disrupt corticogenesis and broader organogenesis. Our multi-tiered in silico–in vitro–in vivo approach demonstrates that noncanonical RAC3 variants can produce complex, multisystem developmental phenotypes beyond previously recognized RAC3-related neurodevelopmental disorders. Full article

Adipose-derived mesenchymal stem cells (ASCs) have great potential in regenerative medicine due to their abundance and innate multi-lineage differentiation potential. However, the therapeutic efficacy of ASCs is often compromised by poor microenvironmental conditions in the damaged tissues after transplantation. In this study, we generated and assessed genetically modified ASCs that expressed granulocyte chemotactic protein-2 (GCP-2) and platelet-derived growth factor-β (PDGF-β). The results revealed that three-dimensional (3D)-cultured ASCs overexpressing GCP-2 and PDGF-β (3D-A/GP) yielded a significant increase in proangiogenic gene expression, cell migration, and endothelial tube formation in vitro. Moreover, the Matrigel plug assay revealed that 3D-A/GP formed functional blood vessels, and 3D-A/GP injection in a hind limb ischemia (HLI) model revealed higher blood flow recovery, limb salvage, and capillary density and lower apoptosis in mice, compared to the controls. Notably, 3D-A/GP exhibited differentiation into endothelial-like cells and upregulated expression of angiogenic factors in ischemic limb tissue. Our results highlight the value of using a combination of genetic engineering and 3D culture systems to improve the therapeutic effect of ASCs in terms of angiogenesis-dependent tissue repair. The dual modulation of GCP-2 and PDGF-β, in combination with 3D culture, presents a new and synergistic opportunity to maximize the use of ASC-based therapies for ischemic diseases and other regenerative medicine applications. Full article

Tumor drug resistance, a major cause of treatment failure, involves complex multi-gene networks, remodeling of signaling pathways, and interactions with the tumor microenvironment. Yin Yang 1 (YY1) is a critical oncogene overexpressed in many tumors and mediates multiple tumor-related processes, such as cell proliferation, metabolic reprogramming, immune evasion, and drug resistance. Notably, YY1 drives resistance through multiple mechanisms, such as upregulation of drug efflux, maintenance of cancer stemness, enhancement of DNA repair capacity, modulation of the tumor microenvironment, and epithelial–mesenchymal transition, thereby positioning it as a pivotal regulator of drug resistance. This review examines the pivotal role of YY1 in resistance, elucidating its molecular mechanisms and clinical relevance. We demonstrate that YY1 inhibition could effectively reverse drug resistance and restore therapeutic sensitivity across various treatment modalities. Importantly, we highlight the promising potential of YY1-targeted strategies, particularly combined with anti-tumor agents, to overcome resistance barriers. Furthermore, we discuss critical translational considerations for advancing these combinatorial approaches into clinical practice. Full article

Background: Boiling histotripsy (BH) uses high-amplitude, short-pulse focused ultrasound to disrupt tissue mechanically. Oncolytic virotherapy using reovirus has shown modest clinical benefit in pancreatic cancer patients. Here, reovirus and BH were used to treat pancreatic tumours, and their effects on the immune transcriptome of these tumours were characterised. Methods: Orthotopic syngeneic murine pancreatic KPC tumours grown in immune-competent subjects, were allocated to control, reovirus, BH and combined BH and reovirus treatment groups. Acoustic cavitation was monitored using a passive broadband cavitation sensor. Treatment effects were assessed histologically with hematoxylin and eosin staining. Single-cell multi-omics combining whole-transcriptome analysis with the expression of surface-expressed immune proteins was used to assess the effects of treatments on tumoural leukocytes. Results: Acoustic cavitation was detected in all subjects exposed to BH, causing cellular disruption in tumours 6 h after treatment. Distinct cell clusters were identified in the pancreatic tumours 24 h post-treatment. These included neutrophils and cytotoxic T cells overexpressing genes associated with an N2-like and an exhaustion phenotype, respectively. Reovirus decreased macrophages, and BH decreased regulatory T cells compared to controls. The combined treatments increased neutrophils and the ratio of various immune cells to Treg. All treatments overexpressed genes associated with an innate immune response, while ultrasound treatments downregulated genes associated with the transporter associated with antigen processing (TAP) complex. Conclusions: Our results show that the combined BH and reovirus treatments maximise the overexpression of genes associated with the innate immune response compared to that seen with each individual treatment, and illustrate the anti-immune phenotype of key immune cells in the pancreatic tumour microenvironment. Full article

Blue light is a significant environmental cue influencing plant photomorphogenesis and regulating plant growth and development. The COP9 signaling complex (CSN), a multi-subunit protein complex, plays a pivotal role in regulating photomorphogenesis, with CSN2 being identified as a key subunit essential for the assembly and function of the CSN. This study investigated the role of OsCSN2 in rice under blue-light conditions. Utilizing OsCSN2 knockout (KO) mutant plants and transgenic overexpression (OE) lines for wild-type (WT) and mutated versions of OsCSN2, we observed significant suppression of the overall seedling phenotype under blue light, indicating that OsCSN2 acts as a negative regulator of blue light-mediated morphogenesis. Further analysis revealed that exogenous application of gibberellin (GA₃) and the GA synthesis inhibitor paclobutrazol (PAC) modulated seedling elongation in response to blue light, particularly affecting plant height, coleoptile, and first incomplete leaf length without altering root growth. This suggests that OsCSN2 mediates the inhibitory effects of blue light on aboveground development through the gibberellin signaling pathway. On day 9, the analyses of endogenous GA₃ levels combined with Western blotting (WB) and quantitative real-time PCR (qRT-PCR) revealed that OsCSN2 senses blue light signals through cryptochrome 2 (CRY2), influences the expression of COP1 and BBX14, and highlights its role in the photoreceptive signaling pathway. This regulation ultimately influences the degradation of SLR1 within the GA signaling pathway, affecting rice seedling growth and development. Our findings also highlight the differential roles of OsCSN1 and OsCSN2 within the CSN in modulating rice seedling photomorphogenesis, thereby providing new insights into the intricate regulatory mechanisms governing plant responses to blue light. Full article