Search Results (245)

Ammonia exposure can induce oxidative stress in aquatic animals. The p62 protein is a selective autophagy receptor that participates in protein degradation and oxidative stress regulation. In this study, the role of Lv-p62 in the response of Litopenaeus vannamei to ammonia exposure was investigated using RNA interference. The results showed that Lv-p62 expression was significantly induced in the hepatopancreas, gills, and intestine of L. vannamei after ammonia exposure (p < 0.05). Lv-p62 expression peaked at 6 h in the gills and 24 h in the intestine, whereas a biphasic response was observed in the hepatopancreas, with an initial peak at 12 h and a higher second peak at 48 h. In the RNAi experiment, Lv-p62 knockdown altered the expression of antioxidant-related genes (Trx, Gst, and Gpx) in a tissue-specific manner, with Gpx expression being prominently increased in the gills and intestine but not in the hepatopancreas under ammonia exposure. Autophagy-related genes (ATG4 and ATG10) also showed time-dependent and tissue-specific expression changes after Lv-p62 knockdown. The expression of apoptosis-related genes, including caspase 3 and p53, was tissue-specific and was generally lower in the dsRNA-Lv-p62+NH₃ group than in the dsRNA-EGFP+NH₃ group at most time points. Histopathological observations showed that hepatopancreatic acinar vacuolation and structural damage were alleviated, and the hepatopancreatic apoptosis rate was reduced in L. vannamei in the dsRNA-Lv-p62+NH₃ group. These findings suggest that Lv-p62 participates in the response of L. vannamei to ammonia exposure, possibly by regulating antioxidant defense, autophagy-related processes, and apoptosis, thereby affecting hepatopancreatic oxidative damage and tissue injury. Full article

Chitin synthase (CHS) catalyzes the final polymerization step in chitin biosynthesis and is therefore central to cuticle formation in arthropods. In this study, a chitin synthase gene from the swimming crab Portunus trituberculatus (PtCHS) was identified and functionally characterized in relation to epidermal formation and molting. The open reading frame of PtCHS was 4731 bp and encoded a predicted protein of 1576 amino acids belonging to glycosyltransferase family 2. Domain prediction revealed multiple transmembrane helices, a conserved chitin-synthase catalytic region, a coiled-coil region, and the diagnostic EDR, QRRRW, and SWGTRE motifs. Phylogenetic analysis assigned PtCHS to the class A/CHS1 chitin synthase lineage, and two alternative splice variants, designated PtCHS1a and PtCHS1b were detected. PtCHS transcripts were broadly distributed across examined tissues, with comparatively high abundance in the Y-organ, midgut, ovary, and epidermis. During the molting cycle, epidermal PtCHS expression increased during premolt, reached its highest level in postmolt stages, and declined during intermolt. During embryonic development, PtCHS expression remained relatively stable until late embryogenesis and then increased sharply before hatching. RNA interference-mediated knockdown of PtCHS reduced the expression of key chitin-biosynthesis genes, decreased epidermal chitin content, prolonged the molting interval, and was associated with molting failure and increased mortality. Conversely, unilateral eyestalk ablation induced PtCHS and molting-related genes, increased epidermal chitin content, shortened the molting interval, and promoted histological features consistent with enhanced extracellular matrix deposition and epidermal biosynthesis. These findings indicate that PtCHS is indispensable for epidermal chitin deposition and successful molting in P. trituberculatus, and provide a molecular basis for understanding molting regulation in economically important portunid crabs. Full article

Periodontal ligament mesenchymal stem cells (PL-MSCs) are vital for both periodontal regeneration and alveolar bone maintenance, including their turnover during orthodontic therapy. Chronic periodontal inflammation, mainly caused by Gram-negative bacterial lipopolysaccharides (LPS), interferes with osteogenic differentiation and leads to bone loss. Increasing evidence indicates that long non-coding RNAs (lncRNAs) link inflammatory signaling to osteogenic regulation, but their specific role in LPS-driven modulation of PL-MSC osteogenesis is not well understood. The aim of this study was to assess the effects of LPS from two bacterial strains on PL-MSCs differentiation. Human PL-MSCs were cultured under standard stem cell or osteogenic conditions and treated with LPS from Escherichia coli or Porphyromonas gingivalis. Mineralization was assessed using Alizarin Red staining. Osteogenic differentiation was evaluated through immunocytochemical analysis of osteopontin, collagen type 1, osteocalcin, osteonectin, and dentin matrix protein-1 (DMP-1). Expression levels of lncRNAs growth arrest-specific transcript 5 (GAS5), Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), maternally expressed gene 3 (MEG3) and Nuclear Enriched Abundant Transcript 1 (NEAT1) were measured by real-time PCR at 6, 24 and 48 h of LPS exposure. Exposure to E. coli LPS significantly inhibited extracellular matrix mineralization and decreased the expression of key osteogenic markers, indicating impaired osteoblast maturation. In contrast, P. gingivalis LPS caused a partial, dysregulated osteogenic response, marked by increased expression of osteopontin, osteonectin, and dentin matrix protein-1 (DMP-1), but without complete differentiation. LPS types altered lncRNA expression profiles, suggesting that non-coding regulatory networks are involved in inflammation-induced osteogenic dysregulation. Multivariate analyses showed decreased expression of GAS5, MEG3, and MALAT1 in the LPS vs. CTR comparison, decreased COL1A1 in LPS-PG vs. CTR, and increased OSTEOPONTIN in LPS vs. CTR. Differentiation was significantly associated with reduced expression of XIST and NEAT1. Time exerted significant effects on GAS5, MEG3, XIST, and MALAT1, with lower expression at 48 h compared with 6 h, and on COL1A1, which was significantly reduced at both 24 h and 48 h relative to 6 h. Bacterial LPS disrupt osteogenic differentiation of PL-MSCs depending on the species, affecting matrix formation, mineralization, and lncRNA expression. These findings highlight lncRNA-mediated communication between inflammatory signals and osteogenic pathways, providing new insights into the molecular mechanisms of inflammation-related bone remodeling in periodontal disease and orthodontic movements. Targeting lncRNA-regulated pathways could be a promising strategy to enhance periodontal regeneration during inflammation and also ensure optimum outcomes in orthodontic therapy. Full article

Background: Hepatocellular carcinoma (HCC) represents an extremely lethal malignancy on a global scale. The clinical significance and molecular mechanisms of the immune-related gene RFXANK in HCC remain unclear. This study seeks to elucidate the clinical implications and diagnostic utility of RFXANK in HCC, while further exploring its underlying molecular mechanisms. Methods: Expression differences of RFXANK in pan-cancer and HCC were analyzed using the TCGA and GEO (GSE45267) databases. Its diagnostic efficacy was evaluated by Cox regression, Kaplan–Meier survival curves, and ROC curves. Potential functional pathways were explored through GO, KEGG, and GSEA enrichment analyses. The correlation between RFXANK and immune cell infiltration, as well as immune checkpoint molecules, was analyzed using the ssGSEA algorithm and CIBERSORTx. In vitro, siRNA interference was employed to knock down RFXANK expression in Huh-7 and MHCC97H cells. The effects on cell proliferation and RAF1 protein levels were assessed using a CCK-8 assay and Western blot, respectively. Results: RFXANK was significantly overexpressed in HCC tissues and was closely associated with aggressive clinical features, including pathological T stage, histological grade, and AFP levels. Multivariate Cox regression analysis confirmed that RFXANK was an independent risk factor for survival in HCC patients (HR = 1.871). The area under the ROC curve (AUC) was 0.939, demonstrating excellent diagnostic predictive value. Enrichment analysis revealed a significant association with the cell cycle, PPAR signaling pathway, and lipid metabolism pathways. Immune infiltration analysis further revealed that RFXANK expression was significantly positively correlated with Th2 and TFH cells, as well as key immune checkpoint molecules such as PD-1, CTLA4, and LAG3, suggesting distinct features of immune polarization and an inhibitory microenvironment. In vitro cellular experiments demonstrated that knocking down RFXANK significantly inhibited the proliferative capacity of HCC cells and reduced RAF1 protein expression. Conclusions: RFXANK may promote HCC progression by driving a multidimensional proliferation–metabolism–immunity mechanism. RFXANK holds promise as a novel biomarker for diagnostic assessment and a potential therapeutic target for HCC patients. Full article

To explore the function of the period gene (Ec-per) in Exopalaemon carinicauda, we cloned the gene of 4611 bp with a 5′UTR of 201 bp, a 3′UTR of 813 bp, and an ORF of 3597 bp encoding 1198 amino acids. The predicted protein includes two PAS and one PERIOD domain. qPCR analysis revealed that Ec-per was expressed across all tissues tested at different developmental stages and during both embryonic and larval stages. Moreover, Ec-per oscillated rhythmically under different conditions of light-to-dark (L:D) ratios, including continuous darkness (0 L:24 D), where changes in the photoperiod influenced amplitude and phase shifts. The knockdown of Ec-per mRNA significantly reduced the expression of the circadian-related genes timeless (tim) and cryptochrome 1 (cry1) (p < 0.05). This suggests that Ec-per is an endogenous clock gene that may participate in molecular feedback loops and synergistically regulate the circadian rhythms through interacting with tim and cry1. RNA interference of Ec-per also markedly downregulated ecdysone receptor mRNA (p < 0.05), suggesting a positive role in the ovarian development of E. carinicauda. In situ hybridization further demonstrated that Ec-per is involved in oocyte proliferation and the accumulation of exogenous nutrients. This study provides new insights for promoting ovarian development and artificial breeding in crustaceans through optimized light-cycle management. Full article

Background/Objectives: Angelman syndrome is a neurodevelopmental disorder resulting from a deficiency of the maternally inherited UBE3A gene. In mature neurons, UBE3A expression is restricted to the maternal allele due to tissue-specific genomic imprinting, while the paternal allele is silenced in cis by the UBE3A antisense transcript (UBE3A-ATS). To date, numerous strategies have been employed to activate paternal UBE3A expression. In this study, we utilized RNA interference (RNAi) to investigate the downregulation of UBE3A-ATS in mouse primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons. Methods: To induce paternal UBE3A expression, we employed small interfering RNA (siRNA) oligonucleotides (20 mouse candidates and 47 human candidates) and lentiviral short hairpin RNA (LV-shRNA) targeting SNORD115 to suppress UBE3A-ATS expression in both mouse primary neurons and iPSCs. Subsequently, we assessed the expression levels of Angelman syndrome-related neighboring and target genes at the transcript and, where applicable, protein levels. Results: Following treatment with siSnord115 or LV-shSnord115, we observed a reduction in Ube3a-ATS and a corresponding activation of paternal Ube3a RNA and protein expression in both Ube3a^P-YFP/m+ and Ube3a^p+/m− mouse primary neurons. A similar effect was observed upon treatment with LV-shSNORD115s in human iPSC-derived neurons. Conclusions: shRNA-mediated inhibition of Ube3a-ATS by targeting Snord115 effectively restores Ube3a/UBE3A expression in both mouse neurons and human iPSCs. While promising, the mild reduction in Snord116 raises concerns about potential off-target effects. AAV-based delivery of shRNA shows potential, but its translational applicability remains to be evaluated in vivo. Full article

Macrobrachium nipponense is an important commercial freshwater prawn species in China. Since larger individuals command higher market value, there is a pressing need to identify growth-related genes and single-nucleotide polymorphisms (SNPs) to facilitate genetic improvement in this species. Previous studies have suggested a potentially regulatory role of an actin-like protein (ACTL) in the growth of M. nipponense. Therefore, the present study aimed to functionally characterize the role of ACTL in growth and identify growth-associated SNPs within this gene. The open reading frame of Mn-ACTL is 1131 bp, encoding a protein with 377 amino acids. Blastx and phylogenetic analyses indicated that Mn-ACTL shares a close evolutionary relationship with orthologs from Macrobrachium rosenbergii and Palaemon carinicauda. The highest expression level of Mn-ACTL in muscle tissue detected by qPCR suggested its potential involvement in growth regulation. RNA interference experiments showed that prawns injected with dsGFP exhibited larger body sizes than those injected with dsACTL, indicating that knockdown of Mn-ACTL expression inhibits growth performance in M. nipponense. Furthermore, muscle tissue from the dsACTL-injected group displayed looser myofibril packing, visibly eroded areas, and increased sarcomere spacing. Collectively, these results demonstrated that ACTL positively regulates growth in M. nipponense. Additionally, the T allele at locus S28_17149891 and the G allele at locus S28_17145758 were significantly associated with growth traits (p < 0.05). In conclusion, this study confirmed the positive regulatory role of ACTL in growth and identified growth-associated SNPs in M. nipponense, providing valuable insights for breeding new varieties with enhanced growth performance in this species. Full article

Salmonella typhimurium (S.T) infection of the central nervous system (CNS) induces severe inflammation, leading to elevated expression of inducible nitric oxide synthase (iNOS) in microglia. This process catalyzes excessive production of nitric oxide (NO), resulting in irreversible damage to neuronal mitochondria. Asiatic acid (AA) is a small molecule with neuroprotective potential; however, its ability to counteract nerve injury induced by S.T and the underlying mechanisms remain unclear. In this study, we established an S.T-infected mouse model (in vivo) and an S.T-stimulated microglial model using BV-2 cells (in vitro) and employed techniques including immunofluorescence (IF), Western blot, co-immunoprecipitation (Co-IP), and RNA extraction and quantitative reverse transcription PCR (RT-qPCR) to systematically evaluate the protective effects and mechanisms of AA. The results showed that pre-treatment with AA significantly reduced the expression of iNOS and the production of NO caused by S.T infection in mouse hippocampal tissue and BV-2 cells. Mechanistically, AA exerts its effects by inhibiting the upstream Toll-like receptor 2 (TLR2)/Notch and nuclear factor-κB (NF-κB) signaling axis. It interferes with the nuclear translocation of Notch and p65 proteins and their complex formation under S.T stimulation, thereby blocking downstream expression of iNOS and production of NO. This study reveals a novel mechanism by which AA alleviates infection-related neuroinflammation through targeting Notch-p65 interactions, providing a new theoretical basis for its clinical application. Full article

Mitochondrial oxidative phosphorylation serves as a critical driving force in the progression of ovarian cancer. Recent studies have demonstrated that copper induces mitochondrial-dependent programmed cell death by directly binding to the thioacylated components of the tricarboxylic acid (TCA) cycle. The involvement of copper in OXPHOS complex IV, a rate-limiting step in the mitochondrial respiratory chain, suggests that the role of mitochondria in mediating copper-induced cell death can be further elucidated through the study of OXPHOS complex IV. The findings of this study indicate that the cuproptosis process in ovarian cancer, induced by Elesclomol, is associated with mitochondrial complex IV, with LRPPRC identified as a crucial factor. Following Elesclomol treatment of ovarian cancer cells, there was a notable increase in mitochondrial reactive oxygen species (ROS), a significant accumulation of the copper death marker protein DLAT, and a marked decrease in the lipoic acid synthesis-related protein FDX1. Furthermore, the expression levels of copper ion transporters ATP7B and CTR1, which are involved in the assembly and translation of complex IV, as well as the core subunit MTCO1 of complex IV, the copper chaperone protein SCO1, and the interacting protein LRPPRC, were significantly diminished. Inhibition of the IV-stabilizing protein LRPPRC in the ovarian cancer cell lines A2780 and SKOV3 through RNA interference resulted in increased sensitivity to Elesclomol. Concurrently, the expression levels of FDX1, LIAS, LIPT1, SCO1, and MTCO1 decreased significantly. These findings suggest that LRPPRC plays a role in inhibiting the expression of lipoic acid and copper chaperone proteins during Elesclomol-induced copper death in ovarian cancer. This inhibition collectively diminishes the expression and activity changes in complex IV, induces mitochondrial dysfunction, and promotes cuproptosis in ovarian cancer. This study further demonstrates that inhibiting the oxidative phosphorylation complex IV can enhance copper-induced cell death in ovarian cancer. Full article

In higher vertebrates, Tripartite Motif (TRIM) proteins modulate the immune response by coordinating processes related to inflammation such as antiviral restriction, autophagy and inflammasome activation. In fish, TRIM proteins have been reported mainly in cyprinids (e.g., carp—Cyprinus carpio and zebrafish—Danio rerio) and salmonids (i.e., rainbow trout—Oncorhynchus mykiss). However, their molecular mechanisms and functions are still being described in aquatic animals. Thus, our study focused on characterizing novel TRIM proteins involved in the innate immunity of gill cells from rainbow trout (RTgill-W1 and primary cultures) stimulated with lipopolysaccharide (LPS) or polyinosinic–polycytidylic acid (poly I:C). Furthermore, an in vivo experiment with rainbow trout was performed to detect TRIM proteins after the challenge with Flavobacterium psychrophilum (a major bacterial pathogen affecting Chilean salmonid industry). In vitro results showed that OmTRIM25 triggered an LPS-induced expression of pro-inflammatory cytokines such as TNF-α2 and IL-1β. Moreover, in the fish experiment, OmTRIM25 and finTRIM2 were up-regulated in the gills two days post-infection (dpi), whereas IL-1β and TNF-α2 had a higher gene expression at four and six dpi, respectively. To investigate the immunological role of OmTRIM25, a gene silencing strategy using RNA interference (RNAi) was used, confirming the immunomodulatory function of OmTRIM25. Full article

Background: Hepatocellular carcinoma (HCC) remains a major global health challenge with limited therapeutic options. Although RNA interference (RNAi) enables precise gene silencing, its clinical application is restricted by siRNA instability, inefficient cellular uptake, and the requirement for potentially toxic delivery carriers. To address these limitations, a dual-targeted branched siRNA nanostructure (GT-multi-siRNA) was developed to simultaneously silence two HCC-related oncogenes, GP73 and hTERT. Methods: GT-multi-siRNA was synthesized in Escherichia coli and characterized for particle size, stability, Dicer processing efficiency, intracellular retention, and cytotoxicity. Its therapeutic effects were evaluated through gene-silencing assays, proliferation and migration assays in Hep3B cells, and intratumoral administration in a xenograft mouse model. Histopathology and cytokine profiling were conducted to assess biosafety. Results: GT-multi-siRNA formed uniform nanoparticles (50–100 nm) with moderate physicochemical stability and minimal cytotoxicity at concentrations ≤ 200 ng/μL. The nanostructure was efficiently processed by Dicer into functional siRNAs and remained detectable intracellularly for at least 36 h. In Hep3B cells, GT-multi-siRNA reduced GP73 and hTERT mRNA and protein levels by approximately 50%, accompanied by significant inhibition of cell proliferation and migration. In vivo, a single intratumoral dose suppressed tumor growth, while a two-dose regimen markedly limited tumor progression. No liver toxicity was observed, and cytokine analysis showed selective IL-4 upregulation without influencing IL-6 levels. Conclusions: GT-multi-siRNA demonstrates potent dual-gene silencing activity and favorable biosafety, providing a promising RNAi-based therapeutic strategy for targeted HCC treatment. Full article

CRISPR-based transcriptional regulation technologies, including CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), offer precise and programmable control over gene expression, representing a major advance in gene and epigenetic therapy. CRISPRa uses nuclease-inactive Cas proteins fused to transcriptional activators to upregulate target genes, while CRISPRi employs repressor domains for gene silencing. Preclinical studies have demonstrated the efficacy of CRISPRa/i in models of metabolic, neurological, muscular, and oncological diseases. Notably, CRISPRi-based therapies have entered clinical trials for conditions like hepatitis B and muscular dystrophy, showing encouraging safety and efficacy profiles. Despite ongoing challenges related to delivery efficiency, immunogenicity, and off-target activity, innovations in protein engineering and guide RNA design are rapidly enhancing the precision and safety of these technologies. Overall, CRISPRa and CRISPRi are poised to transform the treatment of genetic and epigenetic disorders, with continued optimization expected to accelerate their clinical adoption and broaden their therapeutic impact. Full article

Pollution with heavy metals, such as cadmium (Cd), can significantly affect insect growth, development, and behavior. The midgut is an essential organ for stress response. Chitin synthase-2 (CHS-2) is closely associated with forming the peritrophic membrane (PM). The fourth-instar larvae of Aedes albopictus were exposed to varying concentrations of Cd. The results showed that Cd inhibited chitin synthesis and metabolism-related genes, but thickened the midgut PM, indicating that the larvae could respond to Cd stress through the midgut PM. Silencing CHS-2 by RNA interference resulted more severe vacuolization and malformation of midgut epithelial cells without midgut PM protection. Additionally, there was an intensified redox reaction, upregulated expression of metallothionein (MT) and heat shock proteins 70 (HSP70), and increased activity of antioxidant enzymes at some scattered time points. This study confirms that CHS-2 is involved in oxidative stress induced by Cd exposure by regulating PM formation. This study also contributes to further understanding the resistance mechanism of Ae. albopictus under Cd stress, thereby establishing a theoretical foundation for the future studies of them, which is concerned with the possibility of Ae. albopictus as a water environment detection and the control of Ae. albopictus based on resistance mechanism. Full article

Aphids are sap-sucking pests that cause substantial damage to fruit and fibre crops through direct feeding and transmission of plant viruses. While chemical pesticides remain the primary method of control, their use raises concerns related to human health, environmental contamination, pesticide resistance, and impacts on beneficial insects. As a sustainable alternative, spray-on double-stranded RNA (dsRNA) technology offers a promising approach to induce RNA interference (RNAi) in target pests. For RNAi to be effective against sap-sucking insects like the green peach aphid (Myzus persicae), it is essential to identify genes whose silencing disrupts vital physiological functions. In this study, artificial diet (AD)-based feeding assays were used to evaluate dsRNAs targeting eight genes involved in neural function, osmoregulation, feeding behaviour, and nucleic acid/protein metabolism. dsRNAs were administered individually, in combinations, or as a multi-target stacked construct. After 98 h of feeding, aphid mortality ranged from 14 to 72% (individual targets), 78–85% (combinations), and 54% (stacked construct). Transcript knockdown varied from 6.3% to ~54%, though a consistent correlation with mortality was not always observed. The gene targets and combinatorial dsRNA strategies identified in this study provide a foundation for developing RNAi-based crop protection technologies against M. persicae infestation. Full article

Spinal cord injury (SCI) is a neurological condition often resulting in permanent motor and sensory deficits, for which effective treatments remain limited. RNA interference (RNAi) is a post-transcriptional mechanism of the downregulation of gene expression mediated by small interfering RNAs. RNAi has demonstrated therapeutic efficacy in various neurological disorders, positioning it as a promising yet underexplored therapeutic strategy for SCI. Here, we provide a focused overview of the key pathological processes in SCI, including primary mechanical injury and secondary cascades such as inflammation, mitochondrial dysfunction, excitotoxicity, oxidative stress, multiple forms of cell death, and others. The potential of RNAi to selectively silence genes implicated in these pathological processes, thereby enhancing neuroprotection and functional recovery, is highlighted. We point out that not only protein-coding genes, but non-coding RNAs (ncRNAs) are suitable targets for RNAi. Novel RNAi tools such as CRISPR-Cas13 might revolutionize the field and offer new opportunities for SCI therapy. However, despite all these promising findings, relevant translational studies of RNAi remain scarce. Challenges related to delivery methods, long-term efficacy, and cell-specific targeting must be addressed. Importantly, combining RNAi with other strategies such as cell- or biomaterial-based therapies may enhance therapeutic outcomes. Future investigations should prioritize systematic comparisons of RNAi targets and delivery systems, ideally at single-cell resolution and in different SCI models, to identify the most relevant molecular pathways for clinical translation. Overall, RNAi represents a compelling but still underdeveloped approach for SCI therapy, requiring continued refinement to reach clinical application. Full article