Some plant RNA viruses in the family Betaflexiviridae encode a nucleic acid-binding protein (NABP), which enhances viral infectivity. Until recently, Salvia divinorum RNA virus 1 (SdRV1) was the only known member of the genus Citrivirus to encode a NABP. This study aimed to identify novel Betaflexiviridae genomes encoding NABPs and to investigate their evolutionary origins through comparative and phylogenetic analysis. Publicly available plant transcriptome datasets were screened to identify Betaflexiviridae-like viral sequences. Twelve viral genome contigs corresponding to ten distinct viruses were assembled and annotated. Phylogenetic trees were constructed using replicase and NABP protein sequences. Six putative novel species were identified within the genera Citrivirus, Prunevirus, and a potential new genus. Aquilaria sinensis virus 1 (AquSiV1) encoded a NABP, making it the second known Citrivirus with a putative copy of this gene. Hippophae rhamnoides virus 1 (HipRhV1), which also encoded a NABP, formed a distinct lineage, possibly representing a novel genus. Phylogenetic analysis of NABPs revealed topologies incongruent with the replicase phylogeny, suggesting recurrent, independent acquisition events. These findings expand the known genomic and taxonomic diversity of the genera Citrivirus and Prunevirus, and support the hypothesis that NABP genes have been independently and repeatedly acquired within the family Betaflexiviridae.

Hepatocellular carcinoma (HCC) is notable for its marked molecular heterogeneity and poor response to systemic therapy. Targeted next-generation sequencing (NGS) has facilitated the profiling of somatic alterations in HCC. However, the interpretation of panel-based genomic features such as tumor mutational burden (TMB), copy number alterations, and candidate gene fusions remains challenging, particularly in HBV-endemic Asian populations. To explore the somatic mutation landscape of HCC using a targeted sequencing panel in a regional cohort and to descriptively assess its associations with clinicopathological characteristics. We conducted a 671-gene targeted NGS panel covering approximately 2.5Mb of coding regions on tumor-normal paired samples from 40 patients with HCC and integrated the mutational profiles with clinicopathological data. We analyzed somatic mutations, copy number variation signals, and candidate fusion events. We performed functional enrichment analyses and comparative analyses with the Cancer Genome Atlas (TCGA) cohort. Survival analyses were conducted in an exploratory manner. TP53 was the most frequently mutated gene (55%) and its mutation status was associated with hepatitis B virus infection, higher Edmondson-Steiner grade, and microvascular invasion. Panel-derived TMB values were evaluated descriptively, and exploratory survival analyses suggested potential differences between patient subgroups. Two candidate in-frame fusion signals, KIT-PDGFRA and ROS1-FBXL17, were detected in individual samples based on targeted DNA sequencing. Functional enrichment analyses indicated that mutated genes were mainly associated with cancer-related biological processes. This study provides an exploratory overview of somatic alterations detected by a targeted sequencing panel in a regional HCC cohort. Given the limitations inherent to panel-based analyses and the lack of independent validation, these findings should be interpreted cautiously and primarily serve as a reference for future large-scale and experimentally validated studies aimed at refining precision oncology strategies in HCC.

Adoptive cell therapies employing chimeric antigen receptors (CARs) have achieved remarkable success in hematologic malignancies, yet their efficacy in solid tumors remains limited due to the immunosuppressive tumor microenvironment (TME), which restricts immune cell infiltration and persistence. While CAR-NK cells provide potent cytotoxicity with a favorable safety profile, their tumor penetration is often suboptimal. In contrast, macrophages possess intrinsic tumor-homing and tissue-remodeling abilities that could help overcome these barriers. This study aimed to develop a combinatorial innate-cell immunotherapy integrating nanobody-based mesothelin (MSLN)-targeting CAR-macrophages (CAR-Ms) with CAR-NK cells to remodel the TME and enhance antitumor immunity in pancreatic cancer. Using a validated D3 nanobody CAR construct, CAR-Ms were generated from PMA-differentiated THP-1 monocytes and characterized for CAR expression, M1/M2 polarization, migration, phagocytosis, and cytokine secretion. CAR-NK cell migration and cytotoxicity were evaluated using conditioned media (CM) from CAR-M/tumor co-cultures. Synergistic antitumor activity was assessed in an orthotopic MSLN⁺ pancreatic ductal adenocarcinoma (PDAC) model. D3-CAR-Ms exhibited robust CAR expression (> 90%) and sustained an M1-like phenotype (CD86⁺HLA-DR⁺CD204⁻) even after tumor engagement. Functionally, they displayed enhanced migration and infiltration into MSLN⁺ PANC-1 spheroids, along with increased phagocytic and tumoricidal activity. Upon antigen engagement, CAR-Ms secreted high levels of CXCL9, which promoted CAR-NK chemotaxis, degranulation, and cytotoxicity. Sequential administration of CAR-Ms and CAR-NK cells in vivo led to marked tumor regression and durable responses without systemic toxicity. Combinatorial nanobody-based CAR-M and CAR-NK therapy reprograms the TME and establishes a CXCL9-driven feed-forward loop between macrophages and NK cells, leading to synergistic innate immune activation and potent tumor control. This strategy provides a mechanistically grounded and translationally feasible framework for next-generation CAR-based immunotherapies targeting MSLN-expressing solid tumors.

NVP-BEZ235 is a dual PI3K/mTOR inhibitor with promising antitumor activity in clear cell renal cell carcinoma (ccRCC). However, the mechanisms underlying acquired drug resistance remain incompletely defined. To investigate whether epitranscriptomic regulation-specifically N6-methyladenosine (m6A) modification and its reader YTHDF3-drives reversible, non-genetic resistance to NVP-BEZ235 in ccRCC, and to identify key downstream effector genes. NVP-BEZ235-resistant models were established from 786-O and PTEN-deficient Caki-1ko ccRCC cells via chronic drug exposure. Drug withdrawal assays were performed to evaluate the reversibility of resistance. Expression of m6A regulators was assessed by qRT-PCR and Western blotting. YTHDF3 was silenced using lentiviral shRNA, and changes in NVP-BEZ235 sensitivity were evaluated by CCK-8 and cell-cycle assays. Genome-wide m6A-RIP-seq and RNA-seq were integrated to define transcripts with coordinated changes in m6A methylation and expression, and core causal genes for ccRCC were prioritized by summary-based Mendelian randomization (SMR). YTHDF3-RNA interactions and dynamic m6A changes were validated by RIP-qPCR and MeRIP-qPCR, and the functional role of SYNM was tested by loss-of-function studies. NVP-BEZ235-resistant cells displayed a reversible, nonheritable phenotype, partially regaining drug sensitivity after short-term drug withdrawal. YTHDF3 was selectively upregulated in resistant cells, and its knockdown significantly lowered the IC50 of NVP-BEZ235 and suppressed proliferation. Integrated m6A-RIP-seq/RNA-seq analyses revealed extensive reprogramming of m6A methylation, and SMR identified SYNM as a core m6A-regulated gene associated with increased ccRCC risk. SYNM was overexpressed in ccRCC tissues and positively correlated with YTHDF3 expression. YTHDF3 bound SYNM mRNA in resistant cells, while m6A levels and expression of both YTHDF3 and SYNM were dynamically reversible upon drug withdrawal. Silencing SYNM robustly resensitized resistant cells to NVP-BEZ235. Reversible resistance to NVP-BEZ235 in ccRCC is driven, at least in part, by an m6A-dependent YTHDF3-SYNM axis. Targeting YTHDF3 or SYNM may provide a rational strategy to overcome PI3K/mTOR inhibitor resistance in ccRCC.

Intracerebral hemorrhage (ICH) is a primary non-traumatic parenchymal hemorrhage of the brain with a high mortality and disability rate. Acupuncture has been proved to alleviate neurological deficits after ICH. Our previous study found that UBL4A is up-regulated in the ICH rat treated with acupuncture, and may be a potential molecular target for acupuncture treatment of ICH. However, the molecular mechanism behind UBL4A is unclear. UBL4A knockdown was conducted in ICH rats with acupuncture treatment to explore its role in the therapeutic effects of acupuncture on ICH. In vitro, the effects of UBL4A on heme-induced neuronal injury were evaluated using E18 rat embryonic primary cortical neurons. Proteomic experiments were used to verify the regulatory relationship between UBL4A and MAPK14. UBL4A knockdown has opposite effects to the therapeutic effects of acupuncture on ICH, including increasing corner turn score, brain water content and the number of degenerated and apoptotic neuron in ICH rats. UBL4A knockdown increased the level of ROS, OPA1-S/L, p-MAPK14 and mitochondrial Drp1, and decreased mitochondrial membrane potential, ATP level and MFN2 level in rat brain tissue. In vitro, UBL4A overexpression reduced ROS level and mitochondrial membrane potential and inhibited the activation of the MAPK14/Drp1 signaling pathway. Co-IP verified the binding of UBL4A and MAPK14, and MAPK14 overexpression reversed the effect of UBL4A on hemin-induced neuronal apoptosis and ROS level. Our study confirms that UBL4A is involved in the therapeutic effect of acupuncture on ICH, and affects mitochondrial damage and oxidative stress through MAPK14 ubiquitination.

Defensins, small cationic peptides with strong antimicrobial activity, are key effectors of innate immunity. α-defensins, human neutrophil peptides, are produced primarily by neutrophils and serve as an essential component of the airway defense system against invading pathogens. However, accumulating evidence indicates that α-defensins released from human neutrophils are markedly elevated in various lung diseases, where excessive α-defensins exert cytotoxic effects on epithelial and immune cells. We investigated how α-defensins influence macrophage inflammatory responses and aimed to elucidate the molecular mechanisms underlying α-defensin-induced macrophage activation. Through RNA-seq analysis, we identified key molecules potentially involved in α-defensin-induced inflammatory signaling and validated these candidates using qRT-PCR and western blotting. Our results show that α-defensins significantly upregulate both the gene expression and protein levels of RNF31 in macrophages, leading to enhanced phosphorylation of NF-κB p65 and increased production of pro-inflammatory cytokines. Furthermore, when co-cultured with lung epithelial cells, α-defensin-stimulated macrophages induced NLRP3 expression in epithelial cells, suggesting that macrophage-epithelial crosstalk contributes to α-defensin-driven airway inflammation. Together, our results reveal that α-defensins promote macrophage-driven inflammation through RNF31-dependent NF-κB activation and subsequent macrophage-epithelial communication, providing new insight into the inflammatory mechanisms of lung injury. These findings uncover a previously unrecognized α-defensin-RNF31 signaling pathway that amplifies macrophage-mediated airway inflammation, highlighting RNF31 as a potential therapeutic target for inflammatory lung diseases.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder marked by heterogeneous symptoms and comorbidities, complicating diagnosis and obscuring its neurobiological basis. To investigate molecular mechanisms underlying ADHD, particularly the inattentive subtype, by examining Ctnnβ1 (catenin beta 1) and Itgβ1 (integrin beta 1), key components of the Wnt/β-catenin signaling pathway implicated in neurodevelopmental disorders. Spontaneously hypertensive rats (SHR/NCrl), a validated ADHD model, were assessed for attentional performance through behavioral testing. Hippocampal and peripheral blood expression of Ctnnβ1 and Itgβ1 were quantified. A subset received atomoxetine to evaluate pharmacological effects on behavioral and molecular outcomes. SHR/NCrl rats displayed attentional impairments compared with controls. These deficits were accompanied by elevated hippocampal and peripheral expression of Ctnnβ1 and Itgβ1. Atomoxetine treatment normalized gene expression and improved attentional performance, demonstrating amelioration at both molecular and behavioral levels. Dysregulation of Wnt/β-catenin/integrin signaling may contribute to the ADHD pathophysiology. Concordant upregulation of Ctnnβ1 and Itgβ1 in brain and peripheral blood may support their potential as peripheral biomarkers of ADHD. Responsiveness of these genetic markers to atomoxetine treatment indicates potential value for treatment monitoring and supports targeting this pathway in future therapeutic strategies.

Bladder cancer (BCa) is the most frequently seen malignancy of the urinary tract. However, its molecular mechanisms and therapeutic targets are not well established. This study aims to investigate the mechanism by which tissue specific transplantation antigen P35B (TSTA3) mediates the suppression of epithelial-mesenchymal transition (EMT) in BCa through targeted regulation of lysosome-associated membrane protein 2 (LAMP2) expression via the mitogen-activated protein kinase (MAPK) signaling pathway. Public datasets were analyzed to predict TSTA3 expression and prognosis in BCa. TSTA3 and LAMP2 expression levels were examined in 30 paired BCa and adjacent normal tissues, followed by Pearson correlation analysis of their mRNA levels. TSTA3 expression was quantified in T24, BIU-87 and simian virus 40-immortalized human urothelial cell line-1 (SV-HUC-1). T24 and BIU-87 cells were subjected to TSTA3 knockdown or overexpression. Cell proliferation, migration/invasion (Transwell), apoptosis (flow cytometry), EMT markers (immunofluorescence), and LAMP2/MAPK pathway proteins were evaluated. TSTA3 upregulation was demonstrated in public databases, BCa patient tissues, and cell strains. TSTA3 knockdown in T24 cells substantially suppressed proliferation, colony formation, invasion, migration, and apoptosis while increasing E-cadherin and decreasing Vimentin expression, whereas TSTA3 overexpression in BIU-87 cells promoted malignant phenotypes. TSTA3 and LAMP2 mRNA levels showed a strongly negative correlation in BCa patients. LAMP2 knockdown reversed the tumor-suppressive effects of TSTA3 silencing. Inhibition of the MAPK pathway rescued the functional deterioration of T24 cells caused by TSTA3 overexpression. TSTA3 promotes BCa proliferation, migration, invasion, and EMT by regulating LAMP2 to activate the MAPK pathway.