Search Results (1,775)

Cancer remains a leading cause of mortality worldwide, largely due to dysregulated apoptotic signaling and the persistent activation of oncogenic pathways. However, natural products are a promising source of multi-target anticancer agents. In this study, we investigated the anticancer activity and underlying mechanisms of Clausena harmandiana root extract and its major carbazole alkaloid, 7-methoxyheptaphylline, both in vitro and in vivo. High-Performance Liquid Chromatography (HPLC) chemical fingerprinting confirmed the presence of bioactive coumarins and carbazole alkaloids in the extract. Cytotoxicity assays demonstrated that the extract significantly reduced the viability of human colorectal adenocarcinoma (HT-29), human hepatocellular carcinoma (HepG2), human lung adenocarcinoma (A549–Luc2), and murine Lewis lung carcinoma (3LL–Luc2) cells in a dose- and time-dependent manner. Our mechanistic investigations revealed the activation of JNK signaling, downregulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1), and increased cleaved caspase-3 expression, indicating that mitochondrial apoptosis was induced. Notably, 7-methoxyheptaphylline markedly suppressed STAT3 phosphorylation in a concentration-dependent manner, comparable to the STAT3 inhibitor JSI-124. In a syngeneic 3LL–Luciferase2 lung cancer mouse model, oral administration of C. harmandiana capsules significantly reduced tumor growth and bioluminescence intensity compared with controls. These in vivo findings were consistent with the inhibition of STAT3 signaling and induction of apoptosis observed in vitro. Collectively, our results demonstrate that C. harmandiana exerts broad-spectrum anticancer activity through coordinated modulation of the JNK–STAT3 axis, leading to caspase-dependent apoptosis. These findings highlight its potential as a promising candidate for the development of STAT3-targeted anticancer therapies. Full article

Background: Tumor–stroma interactions play a critical role in renal cell carcinoma (RCC) progression. Cancer-associated fibroblasts (CAFs) are considered key components of the tumor microenvironment; however, their origin remains controversial. This study aimed to determine whether bone marrow-derived mesenchymal stem cells (MSCs) contribute to CAF-like stromal changes and RCC progression. Methods: An orthotopic xenograft mouse model was established using luciferase- and GFP-labeled Caki-1 cells. MSCs labeled with PKH26 were administered intravenously. Tumor growth was evaluated using an in vivo imaging system and tumor volume measurements. Immunohistochemical analyses were performed to assess MSC localization and α-smooth muscle actin (α-SMA) expression. In vitro proliferation and migration assays were conducted using direct and indirect co-culture systems. Results: The intravenous administration of MSCs significantly increased tumor growth and bioluminescence intensity in an orthotopic model. The tumor volumes were significantly larger in the MSC-treated versus control group. An immunofluorescence analysis demonstrated partial co-localization of PKH26-labeled MSCs with α-SMA-positive fibroblast-like cells, suggesting acquisition of CAF-like features. Direct co-culture with MSCs significantly enhanced RCC cell proliferation and migration in vitro, whereas culturing in conditioned medium alone did not produce similar effects. Conclusions: Exogenously administered bone marrow-derived MSCs may be recruited into RCC tissues and acquire CAF-like features through interactions with tumor cells. These findings suggest that stromal–tumor cell interactions within the tumor microenvironment may contribute to RCC progression and represent a potential therapeutic target. Full article

Fruit shape is determined by patterns of cell division and expansion during early development, yet the upstream transcription factors coordinating cell wall dynamics and cytoskeletal organization remain largely unknown. Here, we report that SlbHLH113, a bHLH transcription factor, positively regulates tomato fruit elongation. Overexpression (OE) of SlbHLH113 produced elongated fruits with increased length/width ratio, whereas RNAi lines exhibited flattened fruits. Histological analysis revealed that SlbHLH113 alters columella cell polarity—promoting elongated cell morphology without affecting cell area—and reduces columella–placenta width and locule width, without altering pericarp thickness. Transcriptomic profiling identified 87 differentially expressed genes in OE lines, with enrichment in cell wall-related processes. Notably, a pectate lyase gene (PL5) and an expansin gene (EXT90) were down-regulated, while genes involved in oriented cellulose deposition (COBRA4) and ethylene signaling were up-regulated. Importantly, SlbHLH113 physically interacts with the microtubule-associated protein SlIQD21a, as demonstrated by yeast two-hybrid and luciferase complementation assays. Finally, SlbHLH113 did not affect major nutrient contents in red-ripe fruits. Collectively, our findings identify SlbHLH113 as a novel regulator of tomato fruit shape that might act through cell polarity control, cell wall remodeling, and interaction with a microtubule-associated protein, offering a potential target for improving fruit morphology without compromising nutritional quality. Full article

Hereditary sensorineural hearing loss (SNHL) represents a significant global public health burden. DFNX2, an X-linked form of non-syndromic SNHL, is caused by pathogenic variants in the POU3F4 gene. This study aimed to identify a novel POU3F4 mutation and characterize its functional consequences to elucidate the molecular pathogenesis of DFNX2. A three-generation Chinese family with X-linked deafness was recruited. Targeted next-generation sequencing was used to screen candidate variants, which were validated by Sanger sequencing for co-segregation analysis. Functional assays, including subcellular localization, dual-luciferase reporter assay, Western blotting, and homology modeling, were performed to assess the mutation’s effects. A novel frameshift mutation, c.670_673dupGGTA (p.(Asn225Argfs*2)), was identified and showed complete co-segregation with the deafness phenotype. The mutant protein exhibited cytoplasmic mislocalization, and dual-luciferase assays revealed a severe reduction in transcriptional activation capacity, whereas Western blot confirmed stable expression of the truncated protein. Structural modeling predicted the loss of both the POUS and POUH DNA-binding subdomains. Collectively, this study expands the mutational spectrum of POU3F4 and supports previously reported mechanisms underlying DFNX2 pathogenesis. Full article

Flower color is a key ornamental trait in Nobile-type Dendrobium, yet the molecular basis underlying purple flower formation remains poorly understood. In this study, a white-flowered paternal cultivar and its purple-flowered filial line were used to investigate the regulatory mechanism of purple floral pigmentation. Comparative phenotypic analysis showed that floral color divergence was established early during flower development, and anthocyanidin profiling of full-bloom petals revealed that purple flowers accumulated substantially higher levels of most anthocyanidins than white flowers, with delphinidin- and cyanidin-derived anthocyanidins together accounting for 80.26% and 94.56% of the total anthocyanidins in white- and purple-flowered materials, respectively. Transcriptome profiling identified a total of 21,235 differentially expressed genes (DEGs), with significant enrichment of phenylpropanoid- and flavonoid-related pathways, and MYB transcription factors prominently represented among the candidate regulators across the three comparison groups. Among them, DntMYB1 was identified as a C1 subgroup R2R3-MYB associated with floral pigmentation, and transient overexpression assays in Phalaenopsis hybrid V3 and a Nobile-type Dendrobium hybrid supported its positive role in visible purple pigmentation. By integrating RNA-seq and DAP-seq analyses, we identified 3205 candidate downstream targets of DntMYB1 and established a DntMYB1-centered regulatory module. Among these candidates, one Dnt4CL1 gene and one DntF3′H1 gene were validated as robust direct targets of DntMYB1 through yeast one-hybrid, EMSA, and dual-luciferase assays. These findings suggest that DntMYB1 is associated with purple flower formation by coordinately regulating both upstream precursor metabolism and downstream anthocyanin biosynthesis, providing new insight into the molecular regulation of flower color in Nobile-type Dendrobium and useful candidate genes for ornamental trait improvement. Full article

Soil salinization is a major abiotic stress limiting maize (Zea mays L.) growth and productivity worldwide. Recently, many genes involved in salt stress have been identified. However, the molecular mechanisms underlying salt tolerance in maize remain largely elusive. In this study, we identified a member of the ZmPIRIN family genes, ZmPRN1, acting as a negative regulator in response to salt stress. The expression levels of ZmPRN1 were down-regulated under salt and H₂O₂ treatment. Subcellular localization analysis showed that ZmPRN1 is localized to the chloroplast. Under salt stress, the Zmprn1-Mu mutant exhibited higher survival rates and lower reactive oxygen species (ROS) accumulation compared to wild-type plants. Whereas, ZmPRN1 overexpression lines were more sensitive to salt stress, and had higher ROS levels and lower chlorophyll content than wild-type plants. Transcriptome analysis showed that the differentially expressed genes (DEGs) were mainly involved in the oxidation-reduction process. Furthermore, yeast-two hybrid and split-luciferase complementation assays revealed that ZmPRN1 can interact with the chloroplast NDH complex subunit NDF4, the RING-type E3 ubiquitin ligase RING371, and the auxin-responsive protein IAA27. Collectively, our findings demonstrated that ZmPRN1 negatively regulates salt tolerance in maize by modulating ROS homeostasis, providing a valuable genetic resource for breeding salt-tolerant maize varieties. Full article

Histone lactylation acts as a master regulator in tumor development, but its role in a noncoding RNA (ncRNA) network remains unclear. This study aims to reveal the interaction between H3K18la and the lncRNA-miRNA-mRNA regulatory network in hepatocellular carcinoma (HCC). Transcriptome sequencing and ChIP sequencing were performed in HCC and adjacent normal tissues. Cut&Run and qPCR were used to validate the H3K18la enrichment on LINC02732 and CD44 promoter. Dual luciferase reporter assay, qPCR and Western blotting were used to verify the LINC02732-miR-1291-ASF1B axis. Co-Immunoprecipitation was performed to validate ASF1B recruiting p300. CCK8 and mouse subcutaneous tumor formation were performed to demonstrate this axis promoting HCC. H3K18la enrichment on LINC02732 promoter elevates its expression in both HCC samples and cell lines, therefore enhancing ASF1B expression via sponging miR-1291. Moreover, ASF1B, a histone chaperone, promotes H3K18la by recruiting lactyltransferase p300, forming an ASF1B-H3K18la positive feedback loop. The axis upregulates CD44 expression and promotes HCC in vitro and in vivo. These findings demonstrated the influence of H3K18la on the LINC02732-miR-1291-ASF1B axis and the novel role of ASF1B in histone lactylation by recruiting p300, which together promoted HCC proliferation. Full article

Background/Objectives: Tree peony (Paeonia suffruticosa) has a short flowering period, and its regulatory mechanisms remain poorly understood. These limitations have severely constrained its industrial development. Expansins (EXPs) are key proteins that mediate cell wall loosening associated with petal expansion, yet they remain uncharacterized in tree peony. Methods: This study utilized gene family identification, key gene screening and functional research, as well as regulatory network analysis to reveal the role of the EXP family in the regulation of flower opening. Results: This study presents the first genome-wide identification of 36 PsEXP genes in tree peony, classifying them into four evolutionarily conserved subfamilies (PsEXPA, PsEXPB, PsEXLA, and PsEXLB). Promoter analysis revealed that 28 out of 36 PsEXP promoters contain gibberellin (GA)-responsive elements. Exogenous GA₃ treatment significantly promoted flower opening and upregulated eight PsEXPs, with PsEXPA4-1 showing the highest expression level and promoter GA-responsive element abundance. Subcellular localization confirmed that PsEXPA4-1 was targeted to the cell wall. Overexpression of PsEXPA4-1 in Arabidopsis led to early flowering and enlarged petals, indicating that PsEXPA4-1 had the potential to promote cell expansion, consistent with its proposed role in tree peony flower opening. Mechanistically, we identified a bHLH transcription factor, PsbHLH25, whose expression was induced by GA. Y1H and dual-luciferase assays indicated that PsbHLH25 can bind to the PsEXPA4-1 promoter. Conclusions: This study systematically characterized the EXP gene family in tree peony, revealed PsEXPA4-1 as a key effector downstream of GA promoting flower opening, and discovered PsbHLH25 as a transcriptional activator linking GA signaling to PsEXPA4-1. These findings provided important insights into GA-mediated floral opening and genetic resources for understanding the molecular mechanisms and enabling precise flowering time regulation in tree peony. Full article

The Colorado potato beetle, Leptinotarsa decemlineata, is a major insect pest of potatoes. Our previous studies have demonstrated that two cytochrome P450 monooxygenase (P450s) genes, CYP9Z140 and CYP9AY1, and a uridine diphosphate–glycosyltransferase (UGT) gene, UGT321AP1, play important roles in thiamethoxam resistance to L. decemlineata. However, the related upstream regulatory mechanism remains unclear. In this study, we first monitored the resistance of L. decemlineata field populations to thiamethoxam in Xinjiang to determine the resistance ratios. The predicted results demonstrated that four transcription factors (TFs), CncC/Maf, Abd-B, FoxO, and Ptx1, may bind to the core regions of three gene promoters. The qRT-PCR results revealed that the TFs were significantly upregulated by thiamethoxam and exhibited specific spatiotemporal expression patterns. Dual-luciferase reporter assays indicated that the CncC pathway could regulate the expression of three detoxification genes, whereas Abd-B and FoxO only regulate CYP9Z140 and UGT321AP1 expressions, respectively. Ptx1 could regulate the expression of both CYP9AY1 and UGT321AP1. Furthermore, knockdown of several TFs through RNA interference significantly reduced expression of the corresponding detoxification genes, consistent with the dual-luciferase reporter assay results, and increased the thiamethoxam susceptibility of test adults. These findings aid in gaining a deeper understanding of the transcriptional regulation mechanisms of insecticide resistance in insects. Full article

Intramuscular fat (IMF) is a pivotal determinant of meat quality in yaks (Bos grunniens). While nutritional factors are well-documented, the epigenetic landscape, particularly the transcriptional architecture governed by super-enhancers (SEs), remains largely unexplored in the context of IMF deposition. To investigate SE-associated genes, Chromatin immunoprecipitation sequencing (ChIP-seq) assays using H3K27ac antibodies and RNA-sequencing (RNA-Seq) were conducted on longissimus dorsi (LD) muscle tissues with high and low IMF contents. Integrated multi-omics analysis identified 82 enhancer-associated genes exhibiting significant upregulation in high-IMF samples, with 63 loci characterized as SE-associated. In particular, H3K27ac signal distribution analysis indicated that SEs were distributed across functional regions such as promoters, gene bodies, exons, and introns. Among these SE-related genes, 3-hydroxybutyrate dehydrogenase 1 (BDH1) was further investigated to understand its function and regulatory mechanisms. To address this, overexpression or knockdown experiments were conducted, followed by CCK-8, EdU, Bodipy functional assays, and Real-time quantitative PCR (RT-qPCR) analysis. Functional experiments revealed that BDH1 acts as a key positive regulator of yak preadipocyte differentiation and is a prime SE-associated candidate regulatory gene. Furthermore, dual-luciferase reporter assays were performed to identify its SE region, revealing that the activity of 4 enhancer regions was significantly upregulated. Collectively, these findings implicate SE-associated genes in IMF deposition in yaks, provide a valuable resource for future research, and underscore the functional relevance of BDH1 in this process. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating processes such as assembly, genome packaging, and virion maturation. Despite their biological importance, these interactions remain challenging to study and are relatively underexploited as therapeutic targets. Split reporter systems, based on protein-fragment complementation, provide quantitative platforms to measure PPIs by reconstituting reporter activity when interacting protein partners are brought into proximity. These systems can be applied in vitro and in live cells which enables detection of dynamic and multimeric interactions in physiologically relevant contexts. Major classes of split reporter systems include β-lactamase, alkaline phosphatase, luciferase-based platforms, green fluorescent protein, and horseradish peroxidase. Assay performance depends on factors such as fusion protein stability, expression levels, and reporter kinetics, which influence sensitivity, dynamic range, and reliability. These approaches have been applied to study viral protein interactions across diverse systems, including HIV-1 matrix and nucleocapsid proteins, flaviviral capsid proteins, hepatitis B virus core protein, and chikungunya virus capsid. Split reporter assays also enable high-throughput screening for small-molecule inhibitors that disrupt viral PPIs and multimerization. This provides a functional readout linked to viral replication. Despite the challenges that exist in assay optimization and protein stability, the sensitivity and versatility of these systems provide a framework to interrogate viral protein interactions and support the development of antiviral therapeutics.: Full article

Background/Objectives: As a global chromatin organizer, SATB1 is increasingly implicated in neurodevelopmental disorders (NDDs). This study aims to delineate the clinical and molecular characteristics of a novel de novo SATB1 variant in a patient presenting with epilepsy-dominant NDDs phenotypes. Methods: Triggered by the onset of seizures, trio-based whole-exome sequencing (Trio-WES) was performed to identify the genetic etiology. Subsequent sleep electroencephalogram (EEG) and magnetic resonance imaging (MRI) were then conducted to further characterize the patient’s clinical phenotypes. Pathogenicity was assessed through structural modeling and functional characterization. Nonsense-mediated mRNA decay (NMD) status, protein expression profiles, and subcellular localization were determined by reverse-transcription quantitative PCR (RT-qPCR), Western blotting, and immunofluorescence staining. The transcriptional regulatory impacts of the variant were quantified using dual-luciferase reporter system targeting known downstream regulatory elements. Clinical responses to antiepileptic intervention was also monitored. Results: We identified a novel de novo heterozygous pathogenic frameshift variant in SATB1 (NM_002971.5: c.1718_1719insCA; p.Val574Argfs*134) in a patient presenting with early-onset epilepsy, mild intellectual developmental disorder (IDD), speech delay, and dental anomalies. Functional assays demonstrated that the variant-derived transcript escaping NMD, yielding a truncated protein that forms irregular punctate aggregates within nuclei. Dual-luciferase assays revealed significantly increased transcriptional activity, indicating a loss of the protein’s innate transcriptional regulatory capacity. Clinically, treatment with sodium valproate (VPA) successfully stabilized seizures of the patient, markedly reducing both frequency and intensity. Conclusions: The study reports a novel SATB1 frameshift variant that exerts pathogenicity significant functional impairment by disrupting protein localization and transcriptional regulation. These findings expand the genetic spectrum of SATB1-related NDDs and underscore the efficacy of targeted antiepileptic management in genetic diseases. Full article

Metamorphosis in insects is regulated by hormonal signals and metabolic pathways. In this study, we characterized a 20E-mediated miR-2788/Treh1 regulatory axis involved in the larval-to-pupal transition in the leaf beetle Galeruca daurica. Dual-luciferase assays and expression profiling suggest that miR-2788 directly targets and suppresses trehalase 1 (Treh1), a key enzyme for trehalose hydrolysis. We found that exogenous 20E treatment significantly downregulated miR-2788, leading to the derepression of Treh1 and increased expression of chitin biosynthetic genes (CHI, CHS, and GPI). Conversely, miR-2788 overexpression or Treh1 silencing via RNAi resulted in massive trehalose accumulation, reduced chitin synthesis, and lethal developmental arrest during the metamorphic transition. Furthermore, pharmacological inhibition of Treh1 with Validamycin not only recapitulated these chitin-deficiency phenotypes but was also associated with an upregulation of miR-2788. These results describe a signaling cascade where 20E modulates the conversion of carbohydrate to chitin through the miR-2788/Treh1 axis. This study advances our understanding of hormonal–miRNA–metabolic crosstalk and identifies miR-2788 as a potential target for RNAi-based biopesticide development. Full article

High-temperature stress severely limits the growth, development, and productivity of tomatoes. Understanding the molecular mechanisms underlying its thermotolerance is crucial for breeding heat-resistant varieties. This study employed a stepwise experimental strategy to systematically elucidate the role of the chlorophyll a/b-binding protein SlCAB3 in tomato thermotolerance. First, a high-temperature responsive transcription factor, SlDREBA4, previously identified in our lab, was used in a yeast two-hybrid screen to identify potential interacting proteins, including SlCAB3. The interaction between SlDREBA4 and SlCAB3 was further validated using tobacco in vivo luciferase complementation imaging (LCI) and in vitro pull-down assays. Subsequently, the expression patterns of SlCAB3 under heat stress were analyzed, and its biological function was further evaluated through overexpression, gene silencing, and knockout experiments. Additionally, reactive oxygen species (ROS) accumulation, antioxidant enzyme activities, chlorophyll content, and the expression of stress-responsive genes were measured to comprehensively assess their physiological and molecular regulatory roles. The results indicate that SlCAB3 encodes a typical chlorophyll a/b-binding protein and is rapidly induced by heat stress. Overexpression of SlCAB3 significantly enhances plant thermotolerance, evidenced by reduced heat damage, increased chlorophyll content, decreased ROS accumulation, elevated antioxidant enzyme activities, and upregulation of antioxidant-related genes. Conversely, silencing SlCAB3 produces opposite effects. Moreover, co-expression of SlCAB3 with SlDREBA4 further improves thermotolerance, accompanied by enhanced expression of heat shock protein-related and antioxidant-related genes. In conclusion, SlCAB3 is a positive regulator of tomato thermotolerance, and the interaction module formed with SlDREBA4 may collectively enhance heat resistance by strengthening antioxidant defense and heat stress response mechanisms. Full article

Maize (Zea mays L.) is a vital global crop whose productivity is severely threatened by abiotic stresses. Epicuticular waxes provide a hydrophobic barrier that protects land plants from environmental stresses. However, the role of key wax biosynthetic enzymes, such as aldehyde decarbonylase CER1, in maize stress adaptation remains unclear. In this study, we performed a functional characterization of ZmCER1 in maize. Our results show that the overexpression of ZmCER1 in both Arabidopsis and maize substantially improved tolerance to these abiotic stresses. Under stress conditions, the transgenic plants displayed better growth performance, elevated activities of antioxidant enzymes, and reduced levels of oxidative damage markers. Additionally, the alkane content—especially that of C29 and C31—was significantly increased in the ZmCER1OE lines. Through a yeast two-hybrid screening (Y2H screening), we identified the peroxisomal membrane protein ZmPEX14 as an interacting partner of ZmCER1, and the interaction was further confirmed by luciferase complementation (LUC) and bimolecular fluorescence complementation (BiFC) assays. We propose a model wherein ZmCER1 enhances stress tolerance not only by reinforcing the cuticular wax barrier but also by potentially regulating reactive oxygen species (ROS) detoxification via association with ZmPEX14. Collectively, our findings establish ZmCER1 as a key regulator of abiotic stress tolerance in maize and a promising candidate for the molecular breeding of stress-resilient crops. Full article