Search Results (235)

Colorectal cancer (CRC) treatment with standard chemotherapeutics such as capecitabine (CAP) and irinotecan (IRT) is frequently limited by toxicity and resistance. To identify novel adjuvant strategies, we investigated the dichloromethane–methanol (DM) Soxhlet extract of the red alga Jania rubens, previously shown to exert intrinsic antiproliferative effects via reactive oxygen species (ROS) induction, inhibition of epithelial–mesenchymal transition (EMT), and suppression of TET enzymes. The present study evaluated the effect of the DM extract in combination with chemotherapeutic agents in HCT-116, Caco-2, and HT-29 colorectal cancer cell lines, assessing its potential to enhance treatment response. Co-treatment with the DM extract significantly enhanced the cytotoxic and anti-migratory effects of CAP and IRT in HCT-116 and Caco-2 cells. Combination treatment also impaired long-term clonogenic survival, suggesting the inhibition of therapy-resistant subpopulations. Flow cytometric analysis (Annexin V-FITC/PI) revealed a dose-dependent increase in apoptosis in HCT-116 cells following DM treatment. Western blot analysis further supported the pro-apoptotic activity of the DM extract by demonstrating reduced BCL-2 protein expression. The extract additionally modulated the mRNA expression levels of cytokines (TNF-α, IL-6, and IL-10), suggesting potential immunomodulatory effects in colorectal cancer cells. Notably, co-administration of IRT and the DM extract enhanced apoptosis, primarily through the downregulation of the anti-apoptotic gene BCL-2. Together, these findings indicate that the Jania rubens DM extract modulates multiple cellular pathways and enhances the response to conventional CRC chemotherapeutic agents. Full article

This article proposes three possible explanations that relate to the process of eukaryotic translation initiation. These explanations suggest mechanisms as to how concentrations of initiation factors (by mass or by posttranslational modification) can influence start site selection, how regulation of translation by 4E-BP (an inhibitor of m⁷G cap-dependent translation) can be explained by “eIF4F disassembly” and how the scanning mechanism might involve initiation factor binding at the 5′ end of the mRNA to provide for apparent unidirectional Brownian movement to locate the initiating AUG. These represent testable models, although the experiments would not be simple. It is hoped that the insights provided will assist researchers in defining the precise steps and mechanisms of the complex initiation process. It is noted that the better this process is understood, the easier it will be to understand how this process is regulated under a wide variety of biological situations, such as nutritional deprivation, heat shock, cell growth, or disease. Additionally, as initiation is the rate-limiting step in translation, a better understanding of this process should also suggest new avenues to treat various diseases, especially conditions of unrestricted growth. Full article

Background: The m⁷G cap structure, which mimics the natural cap of eukaryotic mRNA, is a critical determinant of mRNA vaccine efficacy, safety, and stability. However, its precise quantification remains challenging due to complex impurity profiles and the high physicochemical similarity between the target cap and related impurities. Although liquid chromatography mass spectrometry (LC-MS) is widely employed for this purpose, current methodologies still face significant limitations, including labor-intensive sample preparation, low analytical throughput, poor reproducibility in quantifying low-level impurities, and a lack of universally applicable strategies across diverse mRNA vaccine platforms. Methods: We systematically optimized sample preparation and LC-MS detection workflows. RNase H-mediated cleavage was compared with DNAzymes, guide DNA probes were rationally designed, and thermostable RNase H was introduced for one-step denaturation and cleavage. To establish an accurate, efficient, and universal sample preparation workflow. Chromatographic conditions were optimized using an ion-pairing reagent system to suppress ESI-MS metal adducts. Eliminating sample purification improves recovery, reduces manual handling errors, and boosts assay efficiency. Results: Through optimally designed guide DNA probes, RNase H cleavage specificity reached ≥98% with high cleavage efficiency, offering higher efficiency than DNAzyme. Furthermore, the incorporation of thermostable RNase H enabled a single-step workflow combining high-temperature denaturation and site-specific cleavage, substantially streamlining sample preparation. On the chromatographic side, optimization of the ion-pairing reagent system effectively suppressed metal adduct formation in electrospray ionization mass spectrometry (ESI-MS). This advancement enabled direct injection of the 5′ cap fragments without purification, achieving high-recovery quantification while demonstrating broad compatibility across mainstream LC-MS platforms. The optimized assay reduces the total analytical workflow from 4~6 h to under 1.5 h. Conclusions: Combining high accuracy, robustness, and broad platform compatibility, this method offers a universal, high-throughput analytical solution for mRNA vaccine quality control and continuous process development. Full article

Background/Objectives: N6-methyladenosine (m⁶A) is the most prevalent and functionally significant internal modification within eukaryotic mRNA. While m⁶A is known to be regulated at internal sites by factors such as splice junctions, the mechanisms governing deposition within the cap-proximal region remain poorly understood. This study aims to determine the patterns of m⁶A stoichiometry in cap-proximal regions and to investigate whether the choice of the specific transcription start site (TSS) can affect m⁶A stoichiometry. Methods: We re-analyzed our published single-nucleotide-resolution CROWN-seq data to quantify m⁶A stoichiometry across transcript isoforms with different TSSs, and assessed the relationship between specific TSSs and specific m⁶A sites (“TSS-m⁶A-site pairs”). Results: We established the first single-nucleotide-resolution dataset of m⁶A stoichiometry across the transcriptome in cap proximal regions, including stoichiometry of m⁶A across 5′ isoforms for each gene. We found that m⁶A deposition is markedly inhibited within a narrow cap-proximal region in a distance-sensitive manner. m⁶A sites located close to both 5′ and 3′ exon ends exhibit low methylation due to the overlap between the cap-proximal and 3′ exon-end exclusion zones. Conclusions: We find that the first exon contains a narrow m⁶A exclusion zone at its 5′ end. As a result, cap-proximal m⁶A sites can have different stoichiometries depending on the TSS choice. As the m⁶A site is positioned farther from the TSS, m⁶A stoichiometry increases. These results reveal that TSS switching is a regulatory mechanism for m⁶A stoichiometry in cap-proximal regions and provide a mechanism for fine-tuning gene expression and mRNA fate through isoform-specific m⁶A modification stoichiometry. Full article

Porcine circovirus type 3 (PCV3) capsid protein (Cap) is a key antigen for immunological studies and vaccine development. Different optimized PCV3 ORF2 sequences were used to construct six baculovirus transfer plasmids, with the pOET1.1-based design yielding the highest Cap level. Cap expression was confirmed by Western blot, IPMA and IFA. Recombinant baculovirus amplification was optimized, achieving the highest titer at an MOI of 0.1 with a 72 h harvest to 10^7.5TCID₅₀/0.1 mL, while maximal Cap production was obtained at an MOI of 0.1 with a 96 h harvest. PCV3 Cap virus-like particles (VLPs) were purified by sucrose density-gradient ultracentrifugation and cation-exchange chromatography, and TEM revealed spherical particles of approximately 17–20 nm. In mice, VLP immunization induced increasing antigen-specific IgG from day 14. Immunization increased both IgG1 and IgG2a without a significant difference, and post-immunization serum specifically recognized PCV3-positive passaged PK-15 cells in an indirect immunofluorescence assay. In splenic lymphocytes, IFN-γ, TNF-α, IL-4, and IL-10 mRNA levels were significantly upregulated (p < 0.01). Moreover, pig challenge data supported the protective potential of PCV3 Cap VLPs in the natural host. In our study, Cap assembled into VLPs and induced immune responses, providing a basis for PCV3 subunit vaccine development. Full article

This review summarizes mechanisms regulating mRNA translation under cellular stress and highlights design strategies to improve translation efficiency and stability in the gene therapy of human diseases. mRNA-based therapeutics are emerging as a versatile gene therapy platform enabling transient and controllable expression of therapeutic proteins for the treatment of cancer, genetic disorders, and inflammatory diseases. The efficacy of mRNA-based gene therapy is strongly influenced by sequence design, chemical modifications, and structural features. Evidence shows that rational mRNA engineering can significantly enhance translation efficiency even under stress conditions that impair canonical protein synthesis, as observed in many pathological states. Cellular stress activates regulatory pathways that suppress global translation; however, optimized mRNA constructs can partially bypass these inhibitory mechanisms, enabling sustained protein expression. By improving mRNA stability and resistance to stress-responsive translational control, robust therapeutic protein production can be achieved even in challenging cellular environments. This article was prepared as a narrative review focused on translational regulation mechanisms relevant to therapeutic mRNA design under cellular stress conditions. Literature was collected from PubMed, Google Scholar, and Web of Science using keywords including “mRNA therapeutics,” “cellular stress,” “translation regulation,” “UTR engineering,” and “cap-independent translation.” Studies published mainly between 2010 and 2025 were considered. Original articles and reviews related to stress-responsive translation and therapeutic mRNA optimization were included, while studies outside the scope of translational control and mRNA engineering were excluded. Priority was given to recent and mechanistically relevant publications. Full article

Background: Translational regulation constitutes a critical layer of gene expression control in plants, yet the contribution of endogenous 5′ untranslated regions (5′ UTRs) to translational efficiency remains incompletely defined. While viral and synthetic leader sequences have been widely used to enhance protein production, comparatively few native plant 5′ UTRs have been systematically characterised. The objective of this study was to identify and functionally evaluate endogenous plant 5′ UTR elements that promote translation through post-transcriptional mechanisms. Methods: A 79-nucleotide fragment (CYSTM α) derived from the 5′ UTR of Arabidopsis thaliana CYSTM1 (AT1G05340) was cloned upstream of reporter genes and assessed using dual-luciferase assays in transient expression systems (Nicotiana benthamiana and A. thaliana) and in stable transgenic Arabidopsis lines. Translational activity was further evaluated in monocot wheat germ extract and in Escherichia coli. Transcript abundance was quantified by qRT-PCR. Publicly available ribosome profiling and m⁶A datasets were analysed to assess translational efficiency and RNA modification status. Results: In N. benthamiana and A. thaliana, CYSTM α increases reporter protein production 3–7 fold relative to the control and 30–130% above the benchmark Tobacco Mosaic Virus (TMV) Ω leader, without altering mRNA abundance. The CYSTM α sequence also enhances luciferase translation in monocot wheat germ extract and elevates translation 5-fold in E. coli. CYSTM α contains three motifs that may promote translation, namely three CAA repeats that are associated with translation initiation, an AMAYAA motif that is associated with eIF3 binding, and two N6-adenosine DRACH sites that are associated with cap-independent translation. Additionally, ribosome profiling revealed high translational efficiency (TE = 3.25) of native CYSTM1. Conclusions: CYSTM α represents a compact endogenous 5′ UTR element that enhances translation across multiple experimental systems. These findings expand the repertoire of plant-derived translational enhancers and provide insight into sequence features associated with efficient mRNA translation in plants. Full article

Background: The real-world risk profiles of newly defined steatotic liver disease (SLD) subtypes—MASLD, MetALD, and ALD—remain incompletely described in community settings. Methods: A cross-sectional analysis of 950 health-checkup participants was conducted. SLD (CAP ≥ 248 dB/m) and significant fibrosis (LSM ≥ 7.0 kPa) were evaluated by transient elastography. Associations between alcohol intake, cardiometabolic factors, fibrosis, and gut microbiota (16S rRNA sequencing) were assessed. Results: Among 950 participants, 310 (33%) had SLD (MASLD, n = 222; MetALD, n = 41; ALD, n = 23). Treated as a continuous exposure, higher alcohol intake was significantly correlated with elevated systolic/diastolic blood pressure, triglycerides, AST, and γ-GTP, but inversely correlated with HOMA-IR (all p < 0.05). In multivariable logistic regression adjusting for cardiometabolic factors, BMI was the only independent predictor of fibrosis (adjusted OR 1.22, 95% CI 1.11–1.35, p < 0.01), whereas alcohol intake showed no independent association. Furthermore, microbiota analysis revealed that ALD-related SLD was characterized by significant depletion of Blautia and enrichment of Gemella (FDR q < 0.05) compared to non-SLD controls, indicating an alcohol-associated dysbiosis signature. Conclusions: In early-stage SLD, alcohol intake continuously exacerbates cardiometabolic risk factors, whereas fibrosis is predominantly driven by BMI. These findings support quantitative alcohol/BMI integration for risk stratification, alongside microbiota profiling to detect ALD-related dysbiosis. Full article

Host receptors can detect traces of non-self-pathogenic RNAs within a sea of cellular mRNA molecules. In host cells, mRNA cap methylation occurs in the nucleus, generating Cap1 and Cap2 structures (m⁷GpppNm and m⁷GpppNmNm, respectively). By contrast, alphavirus genomes carry a Cap0 structure (m⁷GpppN), which lacks 2′-O-methylation. This difference in the structure of the host and viral caps serves as a molecular signature that enables discrimination between self and non-self RNAs. Several host immune sensors, such as RIG-I and IFIT1, recognize the alphavirus Cap0 structure and trigger an antiviral response to restrict viral replication. It has been proposed that IFIT1 sequesters aberrant RNAs, preventing their translation by host ribosomes and blocking viral protein synthesis. However, alphaviruses have evolved molecular strategies to circumvent IFIT1-mediated restriction and facilitate infection in mammalian cells. One such strategy involves the folding of a 5′ RNA structure that hides the cap from host immune sensors. This highlights the dynamic interplay between viral evasion tactics and host immune defenses. This review will discuss how specific modifications at the 5′ end of alphavirus RNA modulate host defenses and how a deeper understanding of the virus–host interaction may inform the development of novel vaccine strategies. Full article

Background: Silver nanoparticles (AgNPs) trigger apoptosis in cancer cells, while albumin nanoparticles enable effective drug delivery. This study compares the antitumor and cytotoxic effects of albumin-coated AgNPs (AgNPs-Alb) versus AgNPs on human prostate cancer cell lines. Method: AgNPs-Alb were synthesized and tested against PC3 and LNCaP prostate cancer cell lines. Characterization via Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Ultraviolet-Visible (UV-Vis) spectroscopy confirmed their properties. IC50 values were determined using MTT assay, with apoptosis assessed by Annexin-V/PI staining. DNA cell cycle was analyzed by PI staining. Migration, proliferation, and nuclear morphology were evaluated through scratch-wound, colony-forming, and Hoechst staining assays. Gene expression of Snail, E-cadherin, VEGF-C, VEGF-A, Bcl2, Bax, and P53 was analyzed using real-time PCR. Results: The IC50 values for AgNPs and AgNPs-Alb were 48 μM and 32 μM in PC3 cells, and 110 μM and 95 μM in LNCaP cells, respectively. AgNPs-Alb significantly inhibited PC3 cell migration compared to AgNPs (p < 0.001) and Bicalutamide (p < 0.0001). In both cell lines, AgNPs-Alb significantly reduced colony formation compared to AgNPs and Bicalutamide (p < 0.05). Flow cytometry revealed a higher percentage of apoptotic cells in PC3 with AgNPs-Alb treatment compared to AgNPs and Bicalutamide. In LNCaP cells, AgNPs-Alb induced a significantly higher percentage of Sub-G1 cells. AgNPs-Alb treatment caused greater mRNA suppression of VEGF-A and a higher Bax/Bcl2 ratio in PC3 and LNCaP cells (p < 0.05). Additionally, a significant increase in P53 and E-cadherin, alongside a decrease in VEGF-C expression in LnCAP cells, was observed (p < 0.05). Conclusions: This study suggests that AgNPs-Alb have stronger anticancer and cytotoxic effects compared to AgNPs alone against PCa cell lines and higher effects were observed on PC3 cells compared to LnCAP cells. Full article

Oncometabolites and hypoxia-regulated exosomes orchestrate hypoxia-inducible factor (HIF)–driven macrophage reprogramming across chronic cardiometabolic and oncologic conditions. In type 2 diabetes (T2D) and obesity, regional hypoxia in expanding white adipose tissue (WAT) reconfigures macrophage immunometabolism and chemokine signaling, recruits C-C chemokine receptor 2 (CCR2⁺) monocytes, and skews adipose-tissue macrophages toward M1-like programs that sustain low-grade inflammation and blunt the physiological M1-to-M2 transition during wound repair. In atherosclerotic plaques, lipid-core hypoxia stabilizes HIF-1α, amplifies nuclear factor kappa-light-chain-enhancer of activated B cells/reactive oxygen species (NF-κB/ROS) signaling, increases matrix metalloproteinase-2/-9 (MMP-2/-9) release, and reduces ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux, weakening the fibrous cap. In tumors, poorly perfused niches accumulate lactate and succinate, which act as paracrine cues. Lactate activates PKA/cAMP pathways and promotes immunosuppressive tumor-associated macrophages (TAMs), whereas succinate signals through succinate receptor 1 (SUCNR1) to reinforce HIF-1α–dependent transcription and M2-like programming. In parallel, hypoxia-regulated exosomes deliver microRNAs such as miR-301a-3p, which suppress phosphatase and tensin homolog (PTEN) and activate PI3Kγ, thereby augmenting immunosuppression and programmed death-ligand 1 (PD-L1) expression. Clinically, this hypoxia–oncometabolite–exosome triad links oxygen debt with macrophage state, plaque destabilization, impaired wound repair, and tumor immune escape. Translational entry points include selective HIF-2α inhibition, phosphoinositide 3-kinase gamma (PI3Kγ) blockade, SUCNR1 targeting, and exosome-based miRNA modulation, while a biomarker panel comprising HIF-1α, vascular endothelial growth factor A (VEGF-A), and MMP-9 offers a pragmatic readout of hypoxia burden, macrophage programming, and therapeutic response. We conducted a focused narrative review (PubMed, Scopus, Web of Science; English; 2003–2025), prioritizing mechanistic and translational studies on hypoxia–HIF, lactate/succinate, and hypoxia-regulated exosomes across T2D, atherosclerosis, and cancer. Full article

Circular RNA (circRNA) has emerged as an alternative RNA modality for vaccine development due to its covalently closed structure and enhanced molecular stability compared with linear messenger RNA (mRNA). Following the clinical success of mRNA vaccines, circRNA-based platforms have gained attention in both prophylactic and cancer immunization. Unlike linear mRNA, circRNA relies on cap-independent translation and is commonly produced through in vitro transcription coupled with ribozyme-mediated self-circularization. In prophylactic vaccination, circRNA vaccines have demonstrated sustained antigen availability, robust humoral and cellular immune responses, and flexibility in multivalent designs and targeted delivery strategies that support germinal center reactions and neutralizing antibody generation. In cancer vaccines, circRNA has been applied to tumor-associated antigens, neoantigens, and non-canonical peptides, with a primary focus on inducing potent antigen-specific CD8⁺ T cell immunity and enabling combination immunotherapy approaches. This review summarizes recent applications of circRNA-based vaccines in prophylactic and cancer settings, emphasizing in vitro transcription-compatible self-circularization strategies and discussing how methodological choices in RNA design, translation control, purification, and delivery shape immunological outcomes. Full article

Background: The long-term stability of mRNA-lipid nanoparticles (LNPs), essential for mRNA vaccines and gene therapies, relies on managing physicochemical properties to preserve their integrity and effectiveness through optimized formulation components. This study systematically evaluated LNP formulations with varied compositions, e.g., Dlin-MC3-DMA and ALC-0315 as ionizable lipids, and DMG-PEG2k or ALC-0159 as polyethylene glycol (PEG)-lipids, stored at −80 °C, −20 °C, 5 °C, and 25 °C in Tris buffer (pH 7.4) for 12 months. Methods: Sixteen quality attributes were analyzed, including particle size, mRNA encapsulation, lipid oxidation, and transfection efficiency over different formulations and storage temperatures to mechanistically evaluate the long-term stabilities. Results: Formulations stored at −80 °C and −20 °C retained acceptable stability, while storage at 5 °C caused aggregation, reduced in vivo expression, and mRNA degradation. Storage at 25 °C led to complete loss of transfection within six months. Mechanistic studies identified oxidative and hydrolytic lipid degradation (e.g., DSPC) in ALC-0315 formulations and MC3 N-oxidation with subvisible particulates in MC3-containing LNPs as primary failure modes. Increasing Tris buffer concentration accelerated 5′-cap hydrolysis, emphasizing the importance of a low-ionic-strength buffer for LNP formulations. Conclusions: Findings re-emphasize the necessity of deep-cold storage (≤−20 °C) and optimized formulation components to preserve mRNA–LNP integrity, offering insights for designing next-generation LNPs with improved shelf-life. Full article

African swine fever virus (ASFV) is a highly contagious and lethal double-stranded DNA virus that relies on host cellular translation machinery for replication and immune evasion. The multigene family 110 (MGF110) contains several members with incompletely defined functions. Here, the role of MGF110-7L in host translation regulation was investigated in HEK-293T and PK15 cells. Ribopuromycylation assays demonstrated that MGF110-7L expression resulted in potent, dose- and time-dependent inhibition of nascent polypeptide synthesis. Western blotting revealed a selective reduction in eIF4G1 protein abundance, with no significant changes in eIF4G2, eIF4E, and eIF4A, while eIF4G1 mRNA levels remained unaffected, indicating post-transcriptional regulation. Overexpression of eIF4G1 partially rescued translation suppression. MGF110-7L also decreased eIF4B phosphorylation and activated the PERK/eIF2α pathway, consistent with the induction of endoplasmic reticulum (ER) stress. ER stress promoted stress granule (SG) formation and enhanced eIF4G1 association with the SG marker G3BP1. The inhibitor assays demonstrated that the suppression of eIF2α phosphorylation by ISRIB restored the abundance of eIF4G1 protein. In addition, the downregulation of eIF4G1 was reversed by the inhibition of autophagy using bafilomycin A1, indicating an SG-linked autophagy–lysosome degradation pathway. Co-immunoprecipitation assays confirmed increased eIF4G1-G3BP1 interaction, but no direct binding between MGF110-7L and eIF4G1. This work provides the first experimental evidence that an ASFV protein, MGF110-7L, suppresses cap-dependent translation through SG-mediated autophagic degradation of eIF4G1, thereby revealing a previously unrecognized mechanism of ASFV translational control. These findings not only extend current understanding of ASFV–host interactions but also suggest potential molecular targets for antiviral strategies and rational vaccine design. Full article

Circular RNA (circRNA) has emerged as a promising vector for drug delivery because, unlike linear mRNA, it does not require costly chemical modifications and offers greater stability and sustained expression in cells. Lacking the canonical 5′ cap structure, circRNA relies primarily on internal ribosome entry sites (IRES) to initiate translation, but IRES-mediated initiation is less efficient than cap-dependent translation. To overcome this limitation, we devised a dual-IRES strategy that introduces a second IRES to drive translation of the coding sequence (CDS). By testing several IRES elements known for high translational activity, this study shows that IRESs derived from the EMCV (Encephalomyocarditis virus) family can enhance expression when placed at the 3′ of the CDS, in coordination with the 5′ EMCV-derived IRES. The optimal dual-IRES combinations identified in this study display compatibility with two different coding sequences, offering a useful strategy to enhance circRNA translation. Full article