Search Results (921)

Background/Objectives: Spheroid cultures in Matrigel are routinely used to study cell behaviour in complex 3D settings, thereby generating preclinical models of disease. Ideally, researchers would like to modulate gene expression ‘in situ’ for testing novel gene therapies while conserving the spheroid architecture. Here, we aim to provide an efficient method to transfect small RNAs (such as microRNAs and small interfering RNAs, i.e., siRNAs) into human induced pluripotent stem cell (iPSC)-derived 3D lung spheroids, specifically alveolar type II epithelial cells (iAT2) and basal cell (iBC) spheroids. Methods: Transfection of iAT2 spheroids within 3D Matrigel ‘in situ’, whole spheroids released from Matrigel or spheroids dissociated to single cells was explored via flow cytometry using a fluorescently labelled siRNA. Validation of the transfection method was performed in iAT2 and iBC spheroids using siRNA and miRNA mimics and measurement of specific target expression post-transfection. Results: Maximal delivery of siRNA was achieved in serum-free conditions in whole spheroids released from the Matrigel, followed by whole spheroids ‘in situ’. ‘In situ’ transfection of SFTPC-siRNA led to a 50% reduction in the SFTPC mRNA levels in iAT2 spheroids. Transfection of miR-29c mimic and miR-21 pre-miR into iAT2 and iBC spheroids, respectively, led to significant miRNA overexpression, together with a significant decrease in protein levels of the miR-29 target FOXO3a. Conclusions: This study demonstrates successful transfection of iPSC-derived lung spheroids without disruption of their 3D structure using a simple and feasible approach. Further development of these methods will facilitate functional studies in iPSC-derived spheroids utilizing small RNAs. Full article

Background: Metabolic flexibility, the ability to efficiently switch between fuel sources in response to changing nutrient availability and energy demands, is recognized as a key determinant of metabolic health. In a recent randomized controlled human feeding trial, overweight individuals receiving American black elderberry juice (EBJ) demonstrated improvements in multiple clinical indices of metabolic flexibility, but the mechanisms of action were unexplored. The objective of this study was to utilize RNA sequencing to examine how EBJ modulates the transcriptional response to fasting and feeding, focusing on pathways related to metabolic flexibility. Methods: Overweight or obese adults (BMI > 25 kg/m²) without chronic illnesses were randomized to a 5-week crossover study protocol with two 1-week periods of twice-daily EBJ or placebo (PL) separated by a washout period. RNA sequencing was performed on peripheral blood mononuclear cells from 10 participants to assess transcriptomic responses collected at fasting (pre-meal) and postprandial (120 min post-meal) states during a meal-challenge test. Results: The fasted-to-fed transition for EBJ showed 234 differentially expressed genes following EBJ consumption compared to 59 genes following PL, with 44 genes shared between interventions. EBJ supplementation showed significantly higher enrichment of several metabolic pathways including insulin, FoxO, and PI3K–Akt signaling. KEGG pathway analysis showed 27 significant pathways related to metabolic flexibility compared to 7 for PL. Conclusions: Our findings indicate that short-term elderberry juice consumption may promote metabolic flexibility in overweight adults. Full article

Aging results from the combined effects of oxidative stress, chronic low-grade inflammation, mitochondrial decline, and cellular senescence, which together drive age-related disorders. Natural products ranging from polyphenols and terpenoids to alkaloids, polysaccharides, peptides, and marine metabolites can influence central pathways such as Nrf2/ARE, NF-κB, MAPK, JAK/STAT, AMPK/PGC1-α, mTOR, and SIRT1/FOXO. By doing so, they strengthen antioxidant defenses, temper inflammation, preserve mitochondrial balance, and regulate autophagy. Increasing attention is also being given to synergy, where combinations of bioactives can achieve stronger and more balanced effects than single agents alone. Advances in artificial intelligence are accelerating this discovery process, while greener extraction and smarter delivery systems such as deep eutectic solvents and nanostructured carriers are improving bioavailability and consistency. Together, these developments underscore the promise of natural product-based strategies for healthy aging. Grounded in rigor and reproducibility, this Special Issue aims to inspire translational advances toward healthier and more graceful aging. Full article

Background/Objectives: Some marine plants and algae are known to exert health benefits. However, the long-term effects and underlying mechanisms of these health benefits are still poorly understood. For this reason, we have investigated an extract from the marsh samphire Salicornia europaea for its life-prolonging potential. Methods: We investigated the effect of an aqueous extract of Salicornia europaea (SEE) on the lifespan of several wild-type strains of Drosophila. In addition, we used deficient flies to elucidate the mechanism of the life-prolonging effects. Finally, we comprehensively phenotyped the treated animals. Results: Supplementing a standard diet with SEE extended the lifespan of different Drosophila laboratory strains by up to a third (37% in w¹¹¹⁸ and 19% in yw). A total of 0.05% of SEE were ineffective, whereas 0.2% induced robust lifespan prolongation. This effect was strictly sex-specific, as the SEE application was completely ineffective in males, while prolonging life in females. We found that the body fat content of SEE-treated female flies was lower compared to controls. The extract also positively impacted the lifespan of flies fed a high-fat diet but not a high-sugar diet. SEE exhibited a lipase-inhibitory activity in vitro. Moreover, SEE counteracted aging-associated loss of intestinal barrier integrity. The sex-specific lifespan extensions induced by the SEE entirely depended on functional Tor signaling in the flies. Tissue-specific silencing of the Tor signaling pathway in different cellular compartments of the intestine reduced, but did not altogether abolish, the lifespan-prolonging effect in females. Conclusions: SEE is a promising candidate for a health-promoting intervention, as it induces lifespan-prolonging and anti-obesogenic effects in a sex-specific manner. These effects depend on functional Tor and partially on FoxO signaling. Future studies should identify the active compounds in the extract. Full article

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young people. Despite the advances in multimodality treatment over recent decades through successive prospective clinical trials, improved rates of survival for patients are mainly limited to those with localised RMS without adverse biologic features. Current clinicopathologic prognostic factors include PAX3(7)::FOXO1 fusion status, the site of primary disease, the pre-chemotherapy extent of disease (including microscopic vs. macroscopic residual disease, locoregional nodal involvement and metastatic status), tumour size and patient age. These factors are used to stratify patients into prognostic risk groups that guide treatment intensity and duration. Risk stratification algorithms are evolving, supported by advances in molecular biology and cancer genomics. In this review we focus on the poorest prognostic groups of paediatric-type RMS (i.e., Very High Risk or relapsed/progressive disease). These include patients whose tumours harbour poor biological characteristics such as PAX3(7)::FOXO1 fusion-positive tumours with locoregional nodal involvement and tumours harbouring other poor-risk genetic variants (particularly MYOD1 and TP53 variants); adolescent and young adult patients; newly diagnosed patients with metastatic RMS; and patients with relapsed and refractory disease. Here we aim to describe the clinical characteristics of these patients, outline current standard multimodality treatments in the context of sequential international clinical trials across the major cooperative groups and summarise emerging novel diagnostic and therapeutic approaches. Full article

Muscle atrophy is defined as reductions in muscle size and function and represents a critical concern affecting elderly populations, immobilized patients, and individuals following specific dietary regimens, such as fasting and low-protein diets. This study investigated the protective effects of Stellaria dichotoma root extract and its isolated bioactive compounds during muscle atrophy using both in vitro and in vivo experimental models. First, S. dichotoma root extract prevented dexamethasone (DEX)-induced atrophy in C2C12 myotubes. Through systematic solvent partitioning and resin chromatography, five compounds (1–5) were successfully isolated from the n-butanol fraction. Dichotomine B (2) was identified as the most abundant and bioactive constituent. Treatment with dichotomine B significantly preserved the myotube diameter, enhanced the fusion index, and maintained the myosin heavy chain protein level while suppressing key atrophic biomarkers, including FoxO3a, MuRF-1, and Atrogin-1, in DEX-treated myotubes. Furthermore, dichotomine B (2) reduced proteolysis in serum-free cultured C2C12 myotubes and in mice subjected to 48 h of fasting, preserving muscle mass and strength. These findings suggest that S. dichotoma root extract and its principal compound, dichotomine B (2), have promising therapeutic potential and provide an opportunity to develop novel pharmacological interventions against muscle wasting through suppression of proteolysis pathways. Full article

Oxidative stress (OS) is known to cause severe liver injury in weaning piglets; however, the cellular and molecular mechanisms underlying this process remain poorly understood. In this study, we employed a diquat (DQ)-induced OS model in weanling piglets and performed single-cell transcriptome sequencing of liver tissue to elucidate the key molecular and cellular events involved in OS-induced hepatic damage. First, piglets were treated with 12 mg/kg DQ and the same amount of saline, and the histopathology, biochemical indicators, and single-cell RNA sequencing (scRNA-seq) of piglets were analyzed. Mouse hepatocytes were used to verify the mechanism of differentially expressed genes, including STAT3 knockdown/overexpression, reactive oxygen species (ROS) detection and apoptosis assay. DQ exposure caused significant oxidative damage in the liver of piglets, which was manifested as decreased superoxide dismutase (SOD) activity (p < 0.05), glutathione (GSH) consumption (p < 0.05) and increased malondialdehyde (MDA) (p < 0.05). Cell type-specific responses were revealed by scRNA-seq, with hepatocytes showing the most pronounced transcriptomic alterations (752 genes up-regulated and 918 genes down-regulated). The expression of STAT3 was up-regulated in hepatocytes (p < 0.05) and down-regulated in B cells. The functional enrichment of macrophages involved FOXO/MAPK signaling and OS pathways. In vitro experiments showed that DQ treatment (IC50 = 125.8 μmol/L) led to an increase in ROS content and apoptosis, STAT3 silencing aggravated ROS and apoptosis (p < 0.05), and STAT3 overexpression alleviated ROS and apoptosis (p < 0.05). STAT3 activation increases HO-1 and Bcl-2, while inhibiting Bax and shifting the Bax/Bcl-2 ratio toward cell survival. It has been shown that DQ induces OS and apoptosis in a cell type-dependent manner, in which STAT3 plays a key regulatory role in antioxidant defense and cell survival. Targeting STAT3 may be a therapeutic strategy for DQ-induced hepatotoxicity. Full article

RASSF4 is a key member of the Ras-associated domain family (RASSF) that exhibits dual functionality in tumorigenesis, playing critical yet context-dependent roles in various malignancies. Its expression is epigenetically regulated through promoter hypermethylation, histone modifications, and microRNAs including miR-155 and miR-196a-5p, which directly target its 3′ untranslated region. In most cancers, such as non-small cell lung cancer (NSCLC) and gastric adenocarcinoma (GAC), RASSF4 acts as a tumor suppressor by inhibiting the RAS/MAPK pathway while activating the Hippo signaling cascade, ultimately inducing cell cycle arrest and apoptosis. Conversely, in aRMS, RASSF4 is upregulated by the PAX3-FOXO1 fusion oncoprotein and promotes tumor growth through MST1 inhibition and subsequent YAP activation. This review systematically analyzes current evidence regarding RASSF4’s complex regulatory mechanisms and clinical significance. We propose targeted therapeutic strategies including epigenetic reactivation, gene intervention, and combination therapies. Furthermore, we identify RASSF4 as a promising diagnostic biomarker and therapeutic target based on integrated mechanistic and clinical evidence. Future research should focus on elucidating context-dependent regulatory switches, developing targeted delivery systems, and validating clinical utility through prospective trials. Full article

Pediatric oral rhabdomyosarcoma (RMS) is a rare and aggressive cancer of the head and neck, characterized by a complex and mostly immunosuppressive tumor–immune microenvironment. Unlike adult cancers, pediatric RMS typically exhibits a “cold” immune profile, characterized by minimal T-cell infiltration, a low mutational burden, and resistance to immune checkpoint blockade. The tumor’s location in the oral cavity adds difficulty to treatment because of anatomical and functional limitations. Additionally, the presence of fusion oncogenes, such as PAX3:FOXO1, hampers immunogenicity and treatment response by disrupting antigen presentation and reducing immune cell infiltration. Advances in immuno-oncology have introduced new strategies, including immune checkpoint inhibitors, chimeric antigen receptor (CAR) therapies, cancer vaccines, and oncolytic viruses. However, these approaches face specific challenges in the pediatric population due to developmental immune factors. This narrative review highlights recent findings on the immunobiology of pediatric oral RMS, focusing on tumor–immune interactions and their impact on disease progression and treatment resistance. We reviewed the cellular components of the TIME, the mechanisms of immune evasion, and the expression of immune checkpoints, including PD-L1 and B7-H3. Emerging immunotherapies, including CAR-T, CAR-NK, and CAR-CIK cell therapies; checkpoint inhibitors; oncolytic viruses; and cancer vaccines, are discussed, with an emphasis on their current limitations and potential to transform the pediatric RMS immune landscape. Full article

This study investigates the effects of saline, alkaline, and combined saline–alkaline water environments on the growth, muscle quality, gene expression, and metabolic profiles of largemouth bass (Micropterus salmoides). Juvenile fish were exposed to five water conditions for 60 days: freshwater (FW), saline water (SW, 10 ppt), alkaline water (AW, 15 mmol/L), and two saline–alkaline combinations (SAW-1: 4 ppt + 10 mmol/L; SAW-2: 6 ppt + 15 mmol/L). While growth rate was similar across groups, SAW-2 caused a significant decrease in survival rate and induced notable alterations in muscle texture and fiber structure. Transcriptomic analyses revealed group-specific enrichment of stress-responsive pathways. The FoxO signaling pathway acts as a central regulator of muscle maintenance and energy reallocation. The solute carrier gene slc38a4 and glula (glutamine synthetase), both closely associated with ammonia detoxification via glutamine synthesis and transport, were upregulated under saline–alkaline stress, indicating enhanced capacity for nitrogen metabolism. In addition, two key regulators of muscle remodeling, loc119898415 and tbx18, were significantly upregulated, suggesting a potential chromatin–transcription program underlying compensatory myogenesis and muscle fiber adaptation in response to environmental challenges. Metabolomic profiling showed an accumulation of osmoprotectants (betaine, taurine) in SW and SAW-2 groups, suggesting enhanced stress resistance. Multiomics integration further indicated coordinated regulation between lipid metabolism and insulin signaling, potentially mediated by the FoxO pathway. These results offer practical guidance for improving largemouth bass aquaculture under inland saline–alkaline conditions. Full article

Non-small cell lung cancer (NSCLC) accounts for over 80% of lung cancer cases and remains challenging to treat due to high recurrence and mortality rates. While cisplatin (CDDP) is a first-line chemotherapy for NSCLC, its clinical utility is limited by toxicity, drug resistance, and inadequate tumor suppression. Plant-derived extracts have shown promise as complementary therapies, offering potential benefits including enhanced efficacy and reduced treatment side effects. The aqueous extract of Actinidia eriantha Benth. root (WE-AER) exhibits significant antitumor activity, though its mechanisms in NSCLC remain unclear. This study demonstrates WE-AER’s potent anti-NSCLC effects through multiple mechanisms. In vitro, WE-AER dose-dependently inhibited A549 cell growth (74.74% inhibition, IC50 = 210.38 µg/mL) by inducing apoptosis via TGF-β/FOXO/mTOR pathway modulation. In vivo, WE-AER suppressed tumor growth and angiogenesis while activating immune responses and reducing inflammation in mouse models, with excellent biosafety. These findings elucidate WE-AER’s anticancer mechanisms and support its potential as a novel herbal therapy for NSCLC. Full article

FOXL2 (forkhead box protein L2) is a transcription factor, its function and regulatory mechanism have been mainly studied in mammals; related research on marine invertebrates is still insufficient. It was found that oogenesis was affected, and even a small number of cells resembling spermatogonial morphology appeared in C. farreri ovaries after the FOXL2 was knocked down through RNA interference (RNAi) technology in our laboratory previously. Based on previous research, this paper conducted transcriptome sequencing and differential expression analysis on the ovarian tissues between the experimental group (post-RNAi) and the control group (pre-RNAi) of C. farreri, and used recombinant C. farreri FOXL2 protein for antibody production in Chromatin Immunoprecipitation Sequencing (ChIP seq) experiments to comprehensively analyze the pathways and key genes regulated by FOXL2 during oogenesis. The results showed that in the RNAi experimental group, 389 genes were upregulated, and 1615 genes were downregulated. Among the differentially expressed genes (DEGs), the differential genes related to gender or gonadal development are relatively concentrated in physiological processes such as steroid hormone synthesis, spermatogenesis, gonadal development, and ovarian function maintenance, as well as the FoxO and estrogen signaling pathways. Combining transcriptome and ChIP-seq data, it was found that there were some genes related to sex gonadal development among genes which were directly regulated by FOXL2, such as Wnt4, SIRT1, HSD17B8, GABABR1, KRAS, NOTCH1, HSD11B1, cPLA2, ADCY9, IP3R1, PLCB4, and Wnt1. This study lays the foundation for a deeper understanding of the FOXL2′s specific regulatory mechanism during oogenesis in scallops as a transcription factor. Full article

Testicular development in male animals is a conserved and highly regulated biological process. Investigating the molecular mechanisms underlying testicular development in Junggar Bactrian camels is essential for gaining a deeper understanding of this process in the species. This study selected testicular tissue from the Junggar Bactrian camel at pre-sexual maturity (G3 group, n = 4, 3 years old) and post-sexual maturity (G5 group, n = 4, 5 years old) for whole transcriptome sequencing and bioinformatics analysis. We identified differentially expressed mRNA (DEmRNA), including KPNA2 and LRRC46; differentially expressed LncRNA (DELncRNA), including LOC123613926 and LOC123613624; and differentially expressed miRNA (DEmiRNA), including eca-miR-196a and eca-miR-183. Additionally, we also identified 87 currently unnamed DEmiRNAs, which are of practical value for future research on the Junggar Bactrian camel testicular development and spermatogenesis. GO and KEGG enrichment analyses showed that DERNA are mainly involved in functions and processes such as protein binding (MF), protein import into nucleus (BP), and extracellular space (CC), as well as signaling pathways such as Insulin, FoxO, MAPK, and PI3K-Akt. Subsequently, we predicted some DEmiRNAs and DELncRNAs association with DEmRNAs, and constructed the competitive endogenous RNA (ceRNA) regulatory network. Finally, we randomly selected 10 DERNAs for RT-qPCR validation, and the transcriptome results were consistent with the RT-qPCR results, indicating that the sequencing results were true and reliable. In conclusion, this study analyzed the differential expression of mRNA, LncRNA, and miRNA in Junggar Bactrian camels before and after sexual maturity, providing data references for future studies related to testicular development and spermatogenesis. Full article

We investigated gene regulatory relationships in the neonatal liver to identify potential mechanisms by which maternal vitamin and mineral supplementation during gestation influences developmental programming in heifer calves. Liver transcriptomes were profiled by RNA-Seq in calves collected 30 h after birth from dams fed either a basal diet (CON; n = 6) or the basal diet plus a vitamin and mineral premix (VTM; n = 6; 113 g·heifer⁻¹·d⁻¹), starting 60 days pre-breeding and continuing through gestation. Using DESeq2, we identified 630 DEGs between treatment groups (p ≤ 0.05 and |log2FC| ≥ 0.5). Over-represented pathways included oxidative phosphorylation, AMPK, PI3K/Akt, and FoxO, key regulators of energy homeostasis. We also identified 58 transcription factors (TFs) and 435 differentially connected genes (p ≤ 0.05), suggesting maternal diet induced a regulatory rewiring of TFs and histone deacetylating genes. Gene set enrichment analysis revealed genes positively ranked, such as LRAT and LMBRD1, underlying the vitamin digestion and absorption pathway, indicating coordinated upregulation in the VTM group. Conversely, 23 DEGs associated with the OXPHOS KEGG pathway were downregulated in the VTM calves. Further research should explore whether these transcriptional changes are linked to epigenetic modifications established at birth, persist throughout postnatal development, and potentially contribute to multigenerational inheritance. Full article