Lenvatinib is a first-line treatment option for advanced hepatocellular carcinoma (HCC), but resistance development is a challenge. A comprehensive and in-depth exploration of the molecular mechanisms underlying Lenvatinib resistance may provide novel insights and strategies for enhancing its therapeutic effectiveness. We established Lenvatinib-sensitive (LS) and Lenvatinib-resistant (LR) HCC models both in vitro and in vivo. Dot blot and Western blot analyses confirmed that the m6A methylation levels were elevated in the LR models, while the expression of ALKBH5 was significantly reduced. Lentivirus-mediated transfection of HCC cells was employed to validate the inhibitory role of ALKBH5 in Lenvatinib resistance in HCC. Mechanistically, we integrated sequencing results from mRNA and methylated RNA immunoprecipitation (MeRIP), followed by validation through RNA immunoprecipitation (RIP), MeRIP-qPCR, dual luciferase reporter assays, Liquid chromatography and tandem mass spectrometry (LC-MS/MS), Enzyme-linked immunosorbent assay (ELISA), and animal experiments (including subcutaneous tumor models and HCC orthotopic mouse models). Results confirmed that ALKBH5 deficiency in LR cells elevated m6A methylation of TBX3 mRNA, thereby enhancing its stability. TBX3 bound to the promoter region of CYP27A1, stimulating its transcription and promoting the synthesis of the cholesterol metabolite 27-hydroxycholesterol (27HC). 27HC could inhibit ferroptosis, thereby protecting LR cells from the targeting effects of Lenvatinib. Additionally, T cell-mediated tumor cell killing and flow cytometry assays evaluated that HCC-derived 27HC promoted CD8+ T cell exhaustion. Our findings indicated that the deficiency of ALKBH5 mediated the enhanced synthesis of the cholesterol metabolite 27HC, which in turn inhibited ferroptosis in HCC cells and the cytotoxicity of CD8+ T cells, leading to Lenvatinib resistance in HCC cells. While the specific role of 27HC was strongly supported, the potential contributions of other CYP27A1-derived metabolites to this phenotype remain a possibility.

While chimeric antigen receptor (CAR) T cell therapies have demonstrated therapeutic efficacy against B cell malignancies, widespread implementation of these therapies is hindered by a cumbersome, ex vivo manufacturing process. Delivery of CAR-encoding messenger RNA (mRNA) to endogenous T cells can generate these therapeutic cells in vivo and streamline this manufacturing workflow. To accomplish this, T cell–activating ligands were conjugated to a biodegradable polymeric mRNA nanoparticle to form T cell–targeted particles. By conjugating multiple activating ligands, T cell transfection and stimulation in vitro was increased, and greater T cell transfection and selectivity in vivo was achieved compared to an untargeted particle. These nanoparticles can flexibly encapsulate mRNA cargos and were used to deliver anti-CD19 CAR mRNA in vivo, enabling depletion of 95% of B cells in the peripheral blood and 50% depletion of splenic B cells in healthy mice. These findings regarding nanoparticle tropism and their potential therapeutic efficacy highlight the importance of this nonviral, polymeric platform to address key limitations associated with current CAR T practices.

Efficient in vivo generation of CAR T cells via mRNA-LNP delivery necessitates high-efficiency T cell engineering, yet current systems remain suboptimal. While lymphoid tropism is essential, it is demonstrated that high tropism alone is insufficient for effective in vivo engineering-some highly tropic LNPs frequently exhibit suboptimal T cell transfection. Crucially, intrinsic T cell transfection potency emerges as an orthogonal and equally critical determinant, consistent with the need for both T cell specificity and transfection efficiency. Through screening a tail-varied ionizable lipid library, H3T4 is identified, a citronellol-tailed LNP with enhanced lymphoid tropism and superior intrinsic potency. The CD3-targeted H3T4 LNP (αH3T4) achieves ~85% in vivo efficacy in T cell engineering, outperforming benchmark LNPs by ∼50% (40% vs. 85%). This proposes a new optimization strategy: effective engineering requires balancing potency and tropism, not maximizing tropism alone. In murine CD19 solid tumor models, αH3T4-generated CAR T cells achieved 83% tumor clearance, demonstrating potency-driven efficacy. This work establishes intrinsic transfection potency as a primary tunable parameter for next-generation CAR T platforms.

Intravenous administration of lipid nanoparticles for the delivery of nucleic acid therapeutics remains constrained by passive uptake mechanisms in the liver, often necessitating high doses to achieve meaningful transfection in specific cells of interest. Targeted LNPs (tLNPs) can overcome these challenges by (i) enabling receptor-mediated endocytosis in difficult-to-transfect cells, thereby reducing passive clearance; (ii) increasing the proportion of LNPs reaching their intended target; and (iii) enabling comparable protein expression at lower doses. Here, we provide a step-by-step guide for formulating tLNPs functionalized with whole antibodies or antibody fragments using traditional laboratory equipment. We outline procedures for antibody preparation and labeling (0.5-1 d), antibody-LNP conjugation (1-2 d), tLNP purification and characterization (1 d) and in vivo and ex vivo targeting evaluation (3-4 d). To demonstrate the versatility of this protocol, we validate in vivo targeting to two mouse tissues: we show that anti-platelet endothelial cell adhesion molecule 1 antibody conjugation to lung-tropic LNPs enhances lung transfection by five times compared to nontargeted LNPs, and anti-epidermal growth factor receptor antibody conjugation to liver-tropic LNPs enhances liver transfection by 20 times. We also demonstrate ex vivo targeting to primary human T cells, where anti-CD5 antibody conjugation to LNPs boosts uptake by 4.5 times and significantly increases mRNA transfection. Importantly, this modular strategy is compatible with any LNP formulation or antibody. In outlining these procedures, we seek to deliver a robust and reproducible workflow for the manufacturing of tLNPs, with the ultimate goal of advancing their therapeutic potential and facilitating clinical translation.

BOLL, a highly conserved gene crucial for meiosis in spermatogenesis, is epigenetically regulated through DNA methylation in various species. This study aimed to determine whether BOLL promoter methylation contributes to its downregulation in azoospermic men with hypospermatogenesis (HS) and to explore its potential regulatory association with human spermiogenesis. We conducted pyrosequencing to assess methylation levels at the putative BOLL promoter on testicular samples from azoospermic men with HS versus normal spermatogenesis (NR) and evaluate correlations with spermatogenic severity and gene expression. In silico CpG island (CGI) prediction and luciferase reporter assays were used to confirm methylation-sensitive regulatory regions. Pathway enrichment analysis identified biological processes associated with BOLL. Functional assays using BOLL overexpression in HEK293T cells and GC2 cells, as well as demethylation using 5-Aza-2'-deoxycytidine (5-AZA) in GC2 cells, were conducted to explore downstream transcriptional effects. Eight CpG sites (CpGs) within the BOLL promoter CGI were significantly hypermethylated in HS samples, with seven exhibiting a significant inverse correlation with the spermatogenic score, and three with BOLL transcript levels. The region from - 1434 to + 180 was confirmed as a methylation-sensitive promoter. Gene ontology analysis indicated that spermatid development and differentiation were the top BOLL-associated pathways. BOLL overexpression by plasmid transfection in HEK293T cells and GC2 cells upregulated spermatid-specific genes (TNP2 and GAPDHS), while concurrently suppressing spermatogonia-associated markers (ID4 and PIWIL4). In GC2 cells, 5-AZA treatment enhanced Boll expression and induced downstream spermatid-specific genes, including Prm2, which was not responsive to BOLL overexpression alone. We identified specific BOLL promoter CpGs that are hypermethylated in HS and associated with reduced gene expression. These findings suggest that promoter methylation may contribute to BOLL silencing and impaired spermatid differentiation, supporting its regulatory role in human spermiogenesis.

Osteoarthritis (OA) is a multifactorial degenerative joint disease. Emerging evidence suggests that dysregulation of circular RNAs (circRNAs) may contribute to OA pathogenesis. In this study, we aimed to investigate the potential role and underlying mechanisms of circ_0004662 in chondrocyte dysfunction under inflammatory conditions. Gene expression was silenced or overexpressed in chondrocytes by cell transfection. Bioinformatics analysis, dual-luciferase reporter assays, and RNA pull-down experiments were employed to validate the interaction within the circ_0004662/miR-450b-5p/TCF4 axis. Cell viability, apoptosis, expression of apoptosis-related proteins, and levels of inflammatory cytokines were assessed using CCK-8, flow cytometry, western blotting, and ELISA, respectively. We found that circ_0004662 was significantly upregulated in OA cartilage tissues and in lipopolysaccharide (LPS)-stimulated chondrocytes. Functionally, knockdown of circ_0004662 attenuated LPS-induced chondrocyte injury, as evidenced by improved cell viability, increased anti-apoptotic protein expression, reduced apoptosis rate, decreased pro-apoptotic protein levels, and lower secretion of inflammatory cytokines. Mechanistically, circ_0004662 acted as a sponge for miR-450b-5p, thereby upregulating TCF4 expression. Our findings suggest that circ_0004662 exacerbates inflammatory chondrocyte injury through the miR-450b-5p/TCF4 axis. These results highlight its potential involvement in OA-related inflammation. Key Points • circ_0004662 was obviously upregulated in OA. • circ_0004662 regulated LPS-induced OA chondrocyte injury by miR-450b-5p/TCF4. • circ_0004662/miR-450b-5p/TCF4 was valuable for the diagnosis of OA diagnosis.

The intracellular delivery of macromolecules is a critical process in cell transfection, gene therapy, and cell-based therapeutics. However, existing microfluidics-based mechanoporation strategies are generally limited by the need to redesign device architectures for specific cell types and sizes, making it difficult to accommodate cellular heterogeneity while maintaining high-throughput operation and thereby limiting the translational potential of these architectures. In this work, we present a zero-net mass-flux (ZNMF) jet-based microfluidic delivery platform driven by electromagnetic actuation, in which periodic suction and expulsion through an orifice yield ZNMF over each actuation cycle while imparting momentum to the surrounding fluid. The oscillation of a magnetic composite membrane generates highly controllable microjets within a microchamber that transiently deform the cell membrane, producing short-lived permeable openings that enable the efficient delivery of macromolecules (4 kDa fluorescein isothiocyanate-dextran). Unlike conventional cell squeezing, this approach does not rely on precise dimensional matching between cells and microchannel constrictions, requires no sheath flow assistance, and exhibits high adaptability, high tunability, and reduced clogging risk. In experiments, we achieved efficient delivery across multiple cell types, including HL-60, MS1, and β-TC-6 cells. Under optimized operating conditions (main-channel flow rate of 10 μl/min, excitation frequency of 88.5 Hz, and current of 2.5 A), HL-60 cells exhibited a delivery efficiency of approximately 43% while maintaining a viability above 85%. Notably, effective delivery and high viability were preserved even when the cell diameter differed by more than 40%. Numerical simulations further revealed that the device can instantaneously amplify the dominant elastic stress by more than fourfold within the jet region, thus establishing a highly localized mechanical environment that facilitates transient membrane permeabilization.

Adeno-associated viral vectors (AAVs) are promising tools for gene therapy. However, scaling up the production of AAVs to produce high-quality vectors at high yields for clinical purposes has proven to be challenging. In the present study, we optimized the production process of AAV in a fixed-bed bioreactor using transient transfection in adherent HEK-293T cells. We systematically optimized the key process parameters, namely cell seeding density, cell density at transfection, and DNA-to-cell ratio, based on the yield obtained, starting from a prototype batch, followed by ten batch runs. Here, we packaged a reporter gene (enhanced green fluorescent protein) and a therapeutic gene (lysyl oxidase) into AAV9 capsids as part of our process development program to be applied for future current Good Manufacturing Practices production and clinical trial application. Throughout the experiments, media conditions, transfection processes, and mechanical parameters were kept identical, while monitoring pH, dissolved oxygen, and media glucose concentration during a production process of approximately 10 days. We demonstrate that by optimizing these parameters, the fixed-bed bioreactor was able to support as many as 1.6-2.8 × 106 cells/carrier strip, up to 3 × 109 cells/m2 bioreactor. Through this multivariate optimization process, we increased viral yield by about 7.6-fold (range of 5.7-10.4-fold for the optimized process runs) over the prototype batch. The total AAV vector yield average was 2.3 × 1014 vg (range 1.1 × 1014 vg to 4.95 × 1014 vg), corresponding to an average per cell yield of 1.4 × 105 vg/cell (range 0.85 × 105-2.46 × 105vg/cell). In conclusion, our findings highlight that optimizing process parameters in a fixed-bed bioreactor presents a promising strategy for scalable and cost-effective AAV vector production.

Forsythiaside A ameliorates acetaminophen-induced liver injury by suppressing ferroptosis through activating USP11/SIRT3-mediated mitophagy.

The infection by Human Papillomaviruses (HPV) has long been established as one of the main causes of cervical cancer, with HPV16/HPV18 high-risk types expressing E6/E7 oncoproteins that inhibit p53/pRB tumor suppressor proteins. Therefore, this work focuses on gene therapy, using the innovative minicircle DNA (mcDNA) vector to protect and express the pri-miR-375 in the cancer cell nucleus, ultimately originating the microRNA-375 (miR-375) in the cytoplasm. This miR-375, initially downregulated in cervical cancer cells, can silence HPV E6/E7 transcripts, thereby negatively regulating the expression of these oncoproteins. The mcDNA-pri-miR-375 vector was successfully constructed, biosynthesized in the host cell, extracted, and purified, followed by several in vitro transfection studies in CaSKi cells (HPV16-infected cervical cancer model) to evaluate the mcDNA-pri-miR-375 effect. FITC-stained-mcDNA-pri-miR-375 was present in cancer cell nucleus, confirmed by confocal microscopy. RT-PCR analysis showed a more intense band of miR-375 transcripts, and RT-PCR/ RT-qPCR confirmed that E6/ E7 transcript levels were nearly 80% diminished 24h after CaSki cells transfection. Also, western-blot showed a decreased band intensity for E6/ E7 proteins on transfected cells. Proliferation and cell invasion studies demonstrated growth and migration arrest for CaSki cells throughout 72h. Cell viability study of these cells also revealed a gradual decrease for the same period, while the NHDF cell viability was not affected, indicating specificity towards CaSki. The consequent effect of silencing the E6/ E7 transcripts was also observed through an increase in p53 protein levels (determined by western blot and ELISA) and caspase-3 activity, after 48h of CaSki cell transfection. These results suggest that mcDNA-pri-miR-375 vector has the potential to be further explored in gene therapy for the treatment of HPV-caused cervical cancer.

Association between polymorphisms in AKR1C2 gene and litter size traits in Xiang pigs.

BACKGROUNDAlcoholic liver disease (ALD) is driven by oxidative stress, lipid metabolism, inflammation, and apoptosis. Current therapies lack efficacy in targeting multi-pathway mechanisms. Xing-Pi-Qing-Gan decoction (XPQG) is an improved traditional Chinese medicine designed to alleviate ALD, but its molecular mechanism remains unknown.AIMTo illustrate the therapeutic targets and molecular pathways of XPQG for the treatment of ALD by integrating chemical profiling, network pharmacology, transcriptomics, and experimental verification in vivo and in vitro.METHODSThe components of XPQG were analyzed using ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry. Then, the protective effect of XPQG on ethanol-induced liver injury, especially its regulatory effect on DDIT3 expression and associated Nrf2/HO-1 antioxidant signaling, was investigated through in vivo animal experiments and in vitro cell experiments. A mouse model of ALD was developed, and the mechanism of XPQG was validated through hematoxylin and eosin (H&E) staining, Western blot, and quantitative RT-PCR. In addition, the key role of DDIT3 in XPQG-mediated protection was further verified by siDDIT3 cell transfection technology. Animal experiments with the reactive oxygen species inhibitor N-acetylcysteine (NAC) further validated the mechanism of XPQG to alleviate liver injury by regulating oxidative stress.RESULTSIn ethanol-treated HepG2 cells, XPQG dose-dependently reduced the formation of lipid droplets, inhibited the expression of tumor necrosis factor-α, interleukin-6, interleukin-1β, and alleviated oxidative stress. In mice, XPQG (15.2 g/kg) lowered the liver/body weight ratio, alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase; H&E and Oil Red O demonstrated a reduction in steatosis. Network pharmacology and RNA-seq converged on MAPK signaling, suggesting DDIT3 as a likely key effector in XPQG-mediated protection. DDIT3 knockdown in HepG2 cells attenuated the benefits of XPQG, supporting DDIT3 as a critical effector mechanism in XPQG-mediated protection. The use of NAC further illustrates the correlation of drugs to oxidative stress in disease effects.CONCLUSIONIn summary, the results of the study suggest that XPQG is effective in improving ethanol-induced acute liver injury (ALD). Its mechanism involves the suppression of DDIT3 and the enhancement of Nrf2/HO-1 pathway activity.

Double pH-responsive lipo-xenopeptides combine polar oligoamino acids (OAAs) such as succinoyl tetraethylene pentamine (Stp) with apolar lipoamino fatty acid (LAF) domains. The resulting potent mRNA carriers undergo a profound shift in their polarity upon endosomal protonation. In the current chemical evolution approach, three top-performing xenopeptides based on a U-shaped (LAF2Stp1, LAF4Stp2) or bundle (LAF4Stp1) topology were modified by replacing Stp with a variety of hydrophobized OAAs. Octanol/water distribution (log D) was determined by a novel label-free mass spectrometry-based method. At neutral pH, the new xenopeptide analogues displayed an up to 85-fold higher hydrophobicity (log D7.4 > 2) than Stp-based carriers, with a sharp pH-responsive decrease at endosomal pH (log D5.5 -1). Transfection efficacies were influenced by the amine count of the OAAs, with tetraethylene pentamine outperforming the OAAs with fewer amines. For both topologies, a strong correlation between mRNA expression and endosomal escape, as quantified by calcein release, was found. For U-shaped carriers (LAF/OAA = 2:1), lipophilic OAAs improved endosomal escape and mRNA transfection efficiency. In contrast, for bundle carriers (LAF/OAA = 4:1), lipophilic OAA incorporation did not further enhance endosomal escape or transfection. Mechanistic studies revealed that LAF-xenopeptide polyplexes can adopt both endosomal pH-dependent and pH-independent cytosolic entry mechanisms, with hydrophobization accelerating expression kinetics and enhancing pH-independent entry. Optimized U-shaped xenopeptides were identified with improved mRNA transfection in tumor cells, a dendritic cell line, and low-passage human colon carcinoma cells. In vivo, enhanced mRNA expression was found upon either intramuscular or intravenous administration in several organs and tumor tissue.

Lipid nanoparticles (LNPs) have emerged as a powerful platform for mRNA vaccine delivery, with ionizable lipids playing a pivotal role in enhancing cellular uptake and endosomal escape, thereby improving therapeutic efficacy. In this study, we designed and synthesized 12 novel ionizable lipids via molecular hybridization, incorporating a hydroxyalkylamine headgroup, an ester linkage, and a cis-double bond tail. These lipids were then formulated into LNPs with helper lipids and eGFP or LUC mRNA. Through cell transfection and in vivo imaging experiments, the O2-N5-OLE LNP was identified with superior mRNA delivery capabilities, particularly in immune cells. Subsequently, it was employed to encapsulate the tumor model antigen ovalbumin (OVA) mRNA vaccine, and its preventive effects against mouse melanoma were evaluated. The results demonstrated that O2-N5-OLE LNP@OVA mRNA exhibits superior tumor suppression. Additionally, the OVA mRNA delivered by O2-N5-OLE LNP significantly elevated serum OVA-IgG and IFN-γ levels and activated dendritic cells and CD8+ T lymphocytes, indicating that the vaccine effectively activated both humoral and cellular immunity. Coupled with the excellent biocompatibility of the O2-N5-OLE LNP, these findings highlight its remarkable delivery efficacy and provide a strong experimental foundation for its potential clinical application.

Synthetic mRNA provides a powerful platform to transfer genetic information encoding therapeutic proteins in vivo. However, their applications are limited by their intrinsic instability and insufficient protein yield. Here we report a 40-nt RNA sequence (ARF6.40) that markedly improves mRNA stability and protein bioavailability of synthetic mRNA. ARF6.40, identified from the 3' UTR of the human ARF6 mRNA, contains a novel CU-rich element that interacts with the protein U2AF2. Compared with the broadly used globin 3' UTR, fusing ARF6.40 to the 3' UTR of EGFP mRNA results in markedly increased mRNA half-life and protein level; transfection of cells with SARS-CoV-2 RBD-encoding mRNA or administration of mice with LNP-encapsulated firefly luciferase mRNA fused with ARF6.40 leads to significantly higher and more sustained RBD secretion or luciferase expression in vivo. Together, our study demonstrates the potential of ARF6.40 in mRNA therapeutics and provides new insights into how the expression of ARF6 is regulated.

Background/Objectives: Antigen presenting cells (APCs) and immune cells have unique properties to drive or suppress immune responses. They are therefore key targets for the expression of vaccine antigens or transgene proteins. To better determine the utility of different molecular therapies to modify these cells, mRNA and DNA-based molecular therapy vectors were compared for their ability to genetically modify immune cells after intradermal injections in mice. DNA-based vectors included naked plasmid DNA, plasmid packaged in lipid nanoparticles (LNPs), and replication-defective adenovirus (Ad) vectors. mRNA delivery was mediated by packaging into LNPs like those used in COVID-19 vaccines. Methods: Each vector was used to deliver Cre recombinase into Cre reporter mice whose cells were activated to express green fluorescent protein (GFP) and firefly luciferase after Cre recombination. The mice were injected intradermally (ID) near the base of their tail at a site that drains into the inguinal lymph node. Luciferase activity was imaged in the living mice 1 or 4 days after vector injection. The animals were then euthanized, and luciferase activity was imaged in the draining inguinal lymph node. Cells were prepared from the intradermal injection site and from the draining lymph node to determine which immune cells were genetically modified by phenotyping CD45, CD3, and CD11b GFP-positive cells by flow cytometry. Given that the skin uniquely contains Langerhans dendritic cells, these CD207+ cells were also phenotyped in skin samples and in the draining lymph node. Results: In both the skin and in the draining lymph node, the rank order of luciferase and GFP activation by the vectors were: (1) Ad; (2) mRNA-LNP; (3) DNA-LNP; and (4) naked DNA. Only mRNA-LNP and Ad vectors mediated obvious luciferase activity in the living animals and in the draining lymph nodes by imaging. Notably, both vectors appeared to leak from the ID injection site and not only modify the draining lymph node but also strongly modify the livers of the mice. Naked DNA and DNA-LNP mediated detectable GFP activation in the skin and draining lymph node in some mice, but this activity was low and did not reach statistical significance when compared to PBS-treated animals. mRNA-LNPs and Ad both mediated significant Cre delivery in CD45+, CD3+, CD11b+, and CD207+ immune cells in the skin and in the lymph node, with adenovirus mediating consistently higher levels of expression in all of the tested cells. Conclusions: These data indicate that mRNA-LNP and Ad vectors mediate stronger modification of skin and lymph node immune cells after intradermal injections. Naked DNA and DNA-LNPs were markedly less potent at this activity than the other vectors. These data are consistent with the higher vaccine potency of mRNA-LNP and Ad vectors and suggest that approaches that increase targeting of immune cell subsets may have utility to increase efficacy while also reducing off-target modification of tissues like the liver.

BackgroundVarious associations between adipose tissue and atherosclerosis (AS) have been revealed. This study aims to identify biomarkers in the epididymal adipose tissue of AS mice and to explore their effects on adipose tissue inflammation and adipogenesis.MethodsThe gene expression profiles of epididymal adipose tissue (GSE57659 and GSE76812) were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEG) screened by Limma R package and genes searched by weighted gene correlation network analysis (WGCNA) were performed to classify common genes associated with AS. The Protein-Protein interaction (PPI) network was constructed by Cytoscape software, and hub genes were eventually determined by the Cytohubba plugin. Finally, one of these hub genes was selected. The cell proliferation ability was assessed using the CCK8 assay. Oil Red O staining and Western blot were employed to evaluate the lipid content in adipocytes. The extent of the inflammatory response in adipocytes was determined by Enzyme-Linked Immunosorbent Assay (ELISA).ResultsA total of 125 DEGs were identified between the control group and the atherosclerosis group. Among these, 34 genes were selected based on two key modules identified through WGCNA. Subsequently, five key nodes were identified, namely Capg, Timp1, Lgals3, Agt, and Mmp9. Capg was selected as the primary gene of interest for further investigation. Following the transfection of 3T3-L1 cells with lentivirus, Capg was overexpressed. Capping actin protein, gelsolin like (CAPG) significantly enhanced preadipocyte proliferation, as demonstrated by CCK-8 and upregulated expression of the Cyclin D1. Furthermore, Oil Red O staining revealed a marked elevation in intracellular lipid accumulation upon CAPG overexpression. Western blot analysis showed increased protein levels of PPAR γ and adiponectin. Furthermore, CAPG in 3T3-L1 cells resulted in a marked upregulation of IL-6 and MCP-1.ConclusionCAPG promotes the proliferation and differentiation of adipocyte precursor cells. Additionally, CAPG enhances the inflammatory response in adipocytes, potentially serving as a key molecule mediating obesity-related atherosclerosis.

This study aimed to establish a technical process for detecting nicotinic acetylcholine receptor (nAChR) antibodies using the transfected cell method and evaluate its application in the serological diagnosis of myasthenia gravis (MG), thereby enhancing diagnostic efficiency. Cell transfection technology was used to introduce various nAChR subunit combinations into HEK293 cells for antibody detection. Indirect immunofluorescence (IIF) was utilized to test nAChR antibodies in serum samples from 85 MG patients, and the results were compared for consistency with those of enzyme-linked immunosorbent assay (ELISA).The combination of fetal and adult AChR subunits in transfected cells exhibited the highest sensitivity for detecting serum antibodies in patients with MG. The prepared cell slides demonstrated excellent consistency with the ELISA kit results for 85 MG patients, yielding a Kappa value of 0.769, indicating excellent agreement between the two methods. Co-transfection of multiple AChR subunits successfully generated a cell expressing clustered nAChRs, establishing a highly sensitive detection technique for nAChR antibodies. This technique is invaluable for serological detection of patients with MG, facilitating early disease detection, condition assessment, and therapeutic guidance.

BackgroundDepression is a prevalent mental disorder, and its pathogenesis has not yet been fully elucidated. Circular RNAs (CircRNAs) have exhibit critical regulatory role in depression, while Unc-51-like kinase 1 (ULK1)-mediated autophagy participates in the pathophysiology of the disorder. Nevertheless, the potential involvement of circRNAs in modulating ULK1-mediated autophagy in depression remains unclear.ObjectivesThis study aimed to identify depression-associated circRNAs regulating ULK1-medaited autophagy, and uncover the underlying mechanisms.MethodsThe depression model was established in mice using chronic unpredictable mild stress (CUMS) procedure. RNA sequencing and bioinformatics analysis were carried out to screen signaling pathways, circRNAs and microRNAs (miRNAs) related to depression. Overexpression and knockdown of circRNAs or miRNAs were conducted by lentivirus microinjection and cell transfection. The depression-like phenotypes were assessed through behavioral tests. Western blot, immunofluorescence and transmission electron microscopy (TEM) were employed to evaluated ULK1-mediated autophagy. RNA pull-down and dual-luciferase reporter assay was executed to explore the underlying mechanisms.ResultsCircADAM9 exhibited the most significant downregulation in CUMS mice among the differentially expressed circRNAs. Overexpression of circADAM9 relieved depressive-like behaviors, and promoted ULK1-mediated autophagy in vivo and in vitro. Importantly, circADAM9 sponged miR-302b-3p to regulate Ulk1 gene expression, and miR-302b-3p mimics diminished the behavioral improvements and pro-autophagic effect modulated by circADAM9.ConclusionWe identified circADAM9 as a novel regulator in depression, which activated autophagy via miR-302b-3p/ULK1 axis to improve depressive-like behaviors. Our findings would provide a new potential therapeutic target for the treatment of depression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13062-026-00738-y.

Background: Malva sylvestris (the common mallow) is an herbaceous species widely used in ethnobotanical practices to treat gastrointestinal, hepatic and urinary inflammation. Objectives: Despite these beneficial effects on human health, the antineoplastic potential of this plant has not yet been fully explored. Thus, in the present study, two human colon cancer cell lines (i.e., HCT-116 and Caco-2) were treated with an extract obtained from M. sylvestris flowers (MFE), whose composition in terms of phytochemicals and microRNAs has been recently published by our research group, to explore its potential bioactivity. Methods/Results: MTT and Trypan blue assays demonstrated that MFE reduced tumour cell growth without causing significant cytotoxicity or apoptosis. Following the diphenylboric acid 2-aminoethyl ester-induced fluorescence of some plant metabolites, microscopy analysis proved that MFE components crossed the cell membranes, accumulating into nuclei. Wound assay and transwell tests documented that MFE was also able to reduce cell motility and invasiveness. In both cell lines qPCR experiments demonstrated that MFE caused the over-expression of factors, like VIMENTIN and E-CADHERIN, which negatively influence epithelial–mesenchymal transition in colon cancers. However, the effects of MFE appeared to be time-, dose- and cell type-dependent. In fact, the treatment induced senescence in P53-null Caco-2 cells (i.e., ROS, β-galactosidase and P21WAF1/Cip1) and a premise of differentiation (i.e., P27Kip1) in P53-wild-type HCT-116 cells, also via the CDK2/c-MYC/AKT axis, justifying its antiproliferative property. In parallel, the transfection of tumour cells with pure synthetic miR160b-5p—a microRNA identified in M. sylvestris flowers and predicted to target the human CDK2 transcript—resulted in gene silencing, thereby suggesting its central role in mediating the cross-kingdom effects of MFE on the investigated cancer models. Conclusions: Overall, these findings open new perspectives on the common mallow as a source of potential antimetastatic compounds and on the possible use of its plant microRNAs in the development of gene therapies.