Search Results (467)

In this work, we designed non-viral gene delivery vector systems based on three poly(2-ethyl-2-oxazoline)-ran-poly[2-(3-butenyl)-2-oxazoline] copolymers functionalized by primary, secondary, and tertiary amino groups. The impact of copolymer structure and composition was sought through the examination of basic physicochemical and biological parameters. The complexation ability of copolymers with plasmid DNA was studied by ethidium bromide quenching assay. The polyplex particles size and ζ-potential were determined by dynamic and electrophoretic light scattering. The release ability of copolymers was assessed by competitive displacement of DNA using dextran sulfate. The biological performance of amino-functionalized poly(2-ethyl-2-oxazoline)-ran-poly[2-(3-butenyl)-2-oxazoline] based gene delivery systems was evaluated, and their behavior under various environmental conditions, such as pH and ionic strength, was investigated. Cytotoxicity was assessed in two human lung-derived cell lines, and the ability of the copolymers to mediate plasmid DNA delivery and expression was examined. The resulting polyplex nanoparticles exhibited the ability to release DNA molecules and sensitivity to alterations in pH and ionic strength. All systems showed high biocompatibility and were able to mediate plasmid DNA delivery, resulting in detectable EGFP expression in vitro. The vector properties were found to be driven by a multifactorial interplay among hydrophobic character, thermoresponsive behavior, polymer mobility, charge accessibility, intracellular environmental responsiveness, secondary structure effects, etc. The copolymer bearing primary amino groups displayed a distinct balance between DNA binding and release, characterized by moderate complex stability and enhanced sensitivity to environmental changes. These findings provide mechanistic insight into how amino functionality and polymer structure influence the structure–property–behavior relationships of polyoxazoline-based non-viral gene delivery systems. Full article

Background: Chronic hepatitis B virus (HBV) infection is a major risk factor of hepatocellular carcinoma (HCC), and hepatocyte-derived host factors play important roles in HBV-associated tumor progression. Alpha-1B glycoprotein (A1BG) is a plasma glycoprotein reported to be dysregulated in multiple cancers. In this study, we investigated the functional role of A1BG in HBV-associated HCC progression. Methods: Both the HepG2 and HBV-transfected HepG2 cell lines were used to examine the biological effects of A1BG. A1BG expression was modulated using siRNA and a plasmid vector. A series of functional assays were conducted to assess cell proliferation, apoptosis, stemness, migration, and invasion. RNA microarray analysis and gene set enrichment analysis (GSEA) were performed to identify A1BG-regulated pathways. Results: Functionally, A1BG overexpression suppressed cell proliferation, stemness, migration, invasion, and HBV products while promoting apoptosis in both HepG2 and HBV-transfected HepG2 cells. In contrast, opposite effects were shown in the event of A1BG knockdown. Moreover, A1BG expression was reduced in HBV-associated HCC tissues and correlated with advanced pathological stage and poor prognosis. RNA microarray analysis and GSEA revealed the activation of anti-HBV-related genes and suppression of FGFR1 signaling and the matrix metalloproteinase pathway in A1BG-overexpressing cells. Conclusions: This study provides evidence that A1BG may be a novel host factor associated with the in vitro suppression of HBV replication and HCC progression by modulating pathways related to enhanced antiviral effects, reduced proliferative capacity and stemness, and suppression of EMT. These findings suggest that A1BG is a potential therapeutic target in HBV-related HCC. Full article

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. Full article

Few differentiation-competent models exist to study early intra-nuclear processes of human papillomavirus (HPV) in keratinocytes. Early HPV DNA replication is usually studied by transfecting transformed or tumor-derived cell lines (C33A, HEK293/HEK293T, CIN612). While these lines support episome replication, their transformed state and oncogene expression can confound interpretation, and they do not undergo the normal keratinocyte differentiation required for the HPV life cycle. We therefore evaluated HaCaT, a spontaneously immortalized, non-transformed keratinocyte line with reversible differentiation, as a model for HPV episomal replication. We optimized culture conditions—particularly extracellular calcium—to toggle HaCaT cells between basal-like proliferation and differentiation, and refined transfection parameters to deliver plasmid vectors required for HPV11 episomal replication. HaCaT cells display differentiation-associated morphological changes and keratin marker expression comparable to primary keratinocytes. In transient luciferase-based origin replicon assays, HPV11 plasmid replicons showed origin-dependent replication in both undifferentiated and differentiated HaCaT cells. Because Ca²⁺-driven differentiation rewires keratinocyte nuclear organization, this Ca²⁺-controlled HaCaT system enables evaluation of early viral nuclear processes, including episomal replication and differentiation-associated increases in replication activity, in a nuclear architecture–dependent epithelial context without exogenous viral oncogenes or cellular transformation. Full article

The GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technology) system enables efficient gene stacking within an Agrobacterium T-DNA. Using unidirectional site-specific recombinases and alternating selection markers, it allows precise, sequential assembly of multiple genes directly within an Agrobacterium virulence plasmid. Here, we modified Agrobacterium tumefaciens strain EHA105 to create JGT105 as a GAANTRY recipient and constructed a 15.8 kb T-DNA containing five cargo sequences. We compared the performance of the JGT105 5-stack strain against a conventional binary vector carrying the same cargo sequences in Arabidopsis and potato transformation. The transformation efficiencies were comparable for the GAANTRY strain and the binary vector (potato: 83% vs. 82%; Arabidopsis: 1.73% vs. 1.95%). Single T-DNA insertion frequencies were also similar between the two systems (17.6% for GAANTRY vs. 24.5% for the binary vector construct in potato; 10.3% vs. 18.2% in Arabidopsis, respectively). Notably, the GAANTRY construct had significantly reduced vector backbone transfer in potato (10.0% vs. 26.5%) for the binary vector, whereas rates were higher in Arabidopsis (37.5% vs. 48.9%). These results show that the JGT105 GAANTRY strain is an effective T-DNA delivery system, matching binary vector transformation efficiency while offering lower backbone integration frequency, facilitating the generation of high-quality, multi-gene transgenic plants. Full article

(1) Background: Echinococcosis is a significant zoonotic disease that the World Health Organization (WHO) aims to eliminate by 2050. Current drug-based control faces challenges such as drug resistance, highlighting the urgent need to develop vaccines as a supplementary strategy. Although some progress has been made in the study of intermediate host vaccines using antigens such as Eg95, there is still no commercial vaccine available for the definitive host, canines—which are crucial for transmission—and it is not yet suitable for large-scale use. While vaccine studies targeting the key enzyme lactate dehydrogenase (LDH) in parasite energy metabolism remain scarce, they represent a promising area of potential. (2) Methods: The B cell antigen epitopes of LDH were analyzed, and prokaryotic (pET-28a-EgLDH) and eukaryotic (pVAX1-EgLDH) DNA vaccine expression vectors were constructed. After verifying expression and immunogenicity via qRT-PCR and WB, in vitro validation was performed in 293T cells. Mice were immunized with the vaccine and then challenged with the parasite; blood was collected from the orbital sinus, and IgG levels and cytokines were measured by ELISA. Protective effects were assessed through counting liver cysts and histopathological analysis. (3) Results: We constructed the pVAX1-EgLDH plasmid and immunized Kunming (KM) mice. Compared with the PBS control group, the vaccine group showed an 80.95% reduction in liver cysts (Quil-A group: 19.00%). Histopathological analysis indicated no significant liver damage, although the spleens in the vaccine group were smaller. ELISA results demonstrated an increase in total IgG (p < 0.05), and cytokine analysis showed elevated levels of IL-1 (p < 0.01), IL-4, and IL-10 (p < 0.001), whereas IL-5 and IFN-γ showed no significant changes (p > 0.05). (4) Conclusions: The EgLDH DNA vaccine can elicit a specific immune response and significantly reduce cyst burden, providing theoretical basis and data support for its use as a candidate vaccine for the prevention and control of Echinococcosis. Full article

The convergence of global plastic pollution and antimicrobial resistance crises has intensified concerns about the role of microplastics (MPs) in disseminating antibiotic resistance genes (ARGs) in marine environments. This review synthesizes the mechanistic pathways through which MPs act as vectors for ARG propagation, supported by a bibliometric analysis of 144 studies retrieved from Scopus. MPs possess distinct physicochemical properties such as nanoplastic formation, polymer-specific sorption, weathering-induced oxidation, and additive leachate release that facilitate microbial colonization and biofilm formation. These plastisphere biofilms, enriched with mobile genetic elements including integrons, transposons, and plasmids, promote ARG transfer via conjugation, transformation, and transduction. Environmental modulators like salinity, oxygen, nutrients, pH, UV exposure, and reactive oxygen species further accelerate horizontal gene transfer, while co-selection pressures from heavy metals and antibiotics amplify resistance dissemination. Bibliometric mapping reveals a sharp rise in publications since 2018, with China leading contributions and major research themes centered on horizontal gene transfer, metagenomics, nanoplastics, and biofilm-mediated resistome evolution. Overall, marine MPs substantially intensify ARG spread through complex microbe–plastic–pollutant interactions, posing significant ecological and public health risks. Addressing current gaps, such as limited field validation, underexplored nanoplastic mechanisms, geographic bias, and lack of standardized monitoring, requires harmonized surveillance, omics integration, pollutant mixture modeling, and One Health-based risk assessment to inform global policy interventions. Full article

Carbon dots (CDs) have emerged as a promising non-viral gene delivery vector due to their excellent biocompatibility and tunable surface properties. In this study, four CDs with gradient-positive zeta potentials (7.23 mV, 16.7 mV, 25.3 mV, 34.5 mV) were synthesized via a hydrothermal method. Among these, CDs-3 with an optimal zeta potential of 25.3 mV stood out, exhibiting ultra-low cytotoxicity (cell viability > 80% even at 50 μg/mL) and a transfection efficiency of nearly 100% (for GFP plasmid delivery), significantly outperforming commercial vectors Lipo2000 and PEI. A stable CDs-3/siIhh delivery system was constructed at a mass ratio of 2:1. In vitro evaluations confirmed that CDs-3/siIhh could efficiently regulate the Indian Hedgehog (Ihh) signaling pathway and osteoarthritis (OA)-related markers in both normal and IL-1β-induced inflammatory ATDC5 chondrocytes. Its regulatory effect was significantly superior to that of the commercial Lipo2000/siIhh and PEI/siIhh systems. This consistent “transcription–translation” regulation, combined with the carrier’s safety and excellent cellular internalization capacity in chondrocytes, highlights its potential for OA gene therapy. Collectively, our work develops a novel, safe, and efficient positive-potential CD-based gene delivery vector, providing a promising gene regulatory capacity by leveraging optimized surface charge engineering. Full article

The rise in antimicrobial resistance (AMR) among the most clinically significant bacteria presents a global threat. The coexistence of resistance mechanisms to both carbapenems and colistin is particularly concerning, as these are last-line treatments, specifically reserved for the most challenging infections caused by clinically multidrug-resistant Enterobacterales. Natural aquatic environments have become environmental reservoirs for the transmission of AMR, particularly concerning mechanisms against these two types of critically important drugs. The crucial role of environmental settings as a driving force for the spread and evolution of AMR associated with these drugs is underestimated, and scientific knowledge on this topic is limited. This review aims to fill an important gap in the scientific literature and comprehensively consolidate the available data on carbapenem- and colistin-associated AMR in the aquatic environment. This study provides a comprehensive synthesis of the current knowledge by integrating bibliographic data with a detailed genomic analysis of 278 bacterial genomes sourced from natural waters. It explores the distribution of carbapenemase and mobile colistin resistance (mcr) genes, identifying their hosts, geographical spread, and complex gene–plasmid–host associations. This review distinguishes two critical host groups for genes that provide resistance to last-resort drugs, Enterobacterales and autochthonous aquatic microbiota, highlighting both confirmed and potential interactions between them. Crucially, genomic analysis highlights the alarming co-occurrence of carbapenem and colistin resistance in single cells and on single plasmids, contributing to the spread of multidrug resistance phenotypes. These findings clearly indicate that aquatic environments are not merely passive recipients but active, evolving hubs for high-risk AMR determinants. Future research should focus on the interplay between allochthonous vectors and autochthonous microbiota to better understand the long-term stabilization of carbapenemase and mcr genes. Such efforts, combined with advanced sequencing technologies, are essential to ensure that carbapenems and colistin remain viable treatment options in clinical settings. Full article

Arthropod-borne orthoflaviviruses, including dengue, Zika, Japanese encephalitis, yellow fever and West Nile viruses, pose a significant global public health threat, causing hundreds of millions of infections annually with severe clinical symptoms. However, the lack of effective vaccines and antiviral drugs, coupled with the biosafety risks associated with handling live highly pathogenic strains, hinders progress in antiviral research. Pseudovirus technology, which uses single-round infectious viral particles lacking replication competence, has thus gained prominence as a safe and versatile tool for antiviral research. This review systematically summarizes the construction, optimization, and applications of orthoflavivirus pseudoviruses in antiviral research. The primary construction strategies of orthoflavivirus pseudoviruses rely on multi-plasmid co-transfection of viral replicons and structural protein expression vectors, leveraging the host cell secretory pathway to mimic natural viral assembly and maturation. The core applications of pseudovirus technology are highlighted, including high-throughput screening and detection of neutralizing antibodies, identification of antiviral drugs targeting viral entry or replication, and evaluation of vaccine immunogenicity. Despite these strengths, the approach still faces limitations, such as incomplete simulation of native viral structures and batch-to-batch titer variability, which may affect the physiological relevance of findings. In summary, orthoflavivirus pseudovirus technology has become an essential platform in both basic virology research and translational medicine, providing critical insights and tools in the ongoing fight against arthropod-borne orthoflaviviruses diseases. Full article

Background: Feline panleukopenia virus (FPV) causes acute and frequently fatal disease in cats, underscoring the urgent need for safe, rapidly effective, and scalable vaccines. While virus-like particle (VLP) vaccines are inherently safe and immunogenic, their development is constrained by low yields of recombinant protein in insect cell expression systems. Methods: An optimized baculovirus expression vector system (BEVS) incorporating the hr1-p6.9-p10 transcriptional enhancer and the Ac-ie-01 anti-apoptotic gene was employed to enhance recombinant protein production. VP2 expression levels, viral titers, and hemagglutination activity were quantified using qPCR, SDS-PAGE/Western blotting, transmission electron microscopy (TEM), and functional assays. Immunogenicity and protective efficacy were assessed in both mice and cats through serological analysis, neutralizing antibody detection, and post-challenge clinical monitoring. Results: The optimized BEVS enhanced recombinant protein transcription by 1.5-fold, viral titers by 3.7-fold, and hemagglutination activity by 15-fold. The purified protein self-assembled into uniform 25 nm virus-like particles (VLPs). Immunization elicited earlier responses compared to commercial vaccines. Vaccinated cats maintained normal body temperature, stable leukocyte counts, and minimal viral shedding following FPV challenge. Conclusions: This study validates an enhanced BEVS that effectively overcomes VP2 yield constraints and generates highly immunogenic FPV VLPs. The platform enables rapid-onset protection and offers a scalable strategy for next-generation FPV vaccine development. Full article

Background: The global dissemination of multidrug-resistant (MDR) Salmonella poses a persistent and serious threat to food safety systems. As a leading poultry-exporting country, Thailand requires a comprehensive understanding of how resistance plasmids spread among Salmonella populations within its chicken production chain. Methods: Between March 2023 and February 2024, 223 Salmonella isolates were collected from chicken slaughterhouses and markets in northeastern Thailand. From these, 19 representative MDR Salmonella enterica isolates, selected based on distinct spatiotemporal distributions, underwent whole-genome sequencing. Genomic analyses included sequence typing, core-genome phylogenetics, and screening for antimicrobial resistance genes. Plasmid replicons were identified, and functional annotation was performed using the COG database. Results: Phylogenetic analysis revealed 11 distinct sequence types within the population. Among these, ST1541 and ST50 showed clear evidence of clonal transmission across different production stages, with a notable clustering pattern observed during the winter season. All sequenced isolates exhibited an MDR phenotype. Plasmids were detected in 78.9% of isolates, with conjugative plasmids being the most frequent type (57.9%). The β-lactamase gene bla_TEM-60 was the most prevalent (78.9%) and showed a strong correlation (r ≥ 0.7) with resistance to both ampicillin and cefotaxime. Functional annotation further revealed an abundance of genes involved in carbohydrate and amino acid metabolism across all isolates. Conclusions: These findings indicate that MDR Salmonella dissemination is driven by two synergistic mechanisms: the clonal expansion of fit lineages and the horizontal transfer of conjugative plasmids harboring β-lactamase genes. We identified IncI-gamma-K1 and Col-related plasmids as key vectors in this process. This study advocates for targeted interventions, guided by a One Health approach, that specifically aim to disrupt plasmid transmission at critical control points, such as slaughterhouses, to curb the spread of antimicrobial resistance. Full article

Cancer immunotherapy increasingly relies on nucleic acid-based vaccines, yet achieving efficient and safe delivery remains a critical limitation. Polyplexes—electrostatic complexes of cationic polymers and nucleic acids—have emerged as versatile carriers offering greater chemical tunability and multivalent targeting capacity compared to lipid nanoparticles, with lower immunogenicity than viral vectors. This review summarizes key design principles governing polyplex performance, including polymer chemistry, architecture, and assembly route—emphasizing microfluidic fabrication for improved size control and reproducibility. Mechanistically, effective systems support stepwise delivery: tumor targeting, cellular uptake, endosomal escape (via proton-sponge, membrane fusion, or photochemical disruption), and compartment-specific cargo release. We discuss therapeutic applications spanning plasmid DNA, siRNA, miRNA, mRNA, and CRISPR-based editing, highlighting preclinical data across multiple tumor types and early clinical evidence of on-target knockdown in human cancers. Particular attention is given to physiological barriers and engineering strategies—including size-switching systems, charge-reversal polymers, and tumor-penetrating peptides—that improve intratumoral distribution. However, significant challenges persist, including cationic toxicity, protein corona formation, manufacturing variability, and limited clinical responses to date. Current evidence supports polyplexes as a modular platform complementary to lipid nanoparticles in selected oncology indications, though realizing this potential requires continued optimization alongside rigorous translational development. Full article

The mammary gland is a valuable model in cancer research and developmental biology. Gene delivery techniques are crucial for mammary tissue research to understand how genes function and study on diseases such as cancer. Viral vector-based approaches provide a high degree of transduction efficiency, but they raise safety and immunogenicity concerns, whereas non-viral vector-based approaches are considered safer and have lower immunogenicity than viral methods. Unfortunately, non-viral gene delivery has rarely been applied to the mammary glands because it is technically challenging. Here, we developed a novel method for in vivo transfection of epithelial cells lining murine mammary glands via intraductal injection of plasmid DNA using a breath-controlled glass capillary and subsequent electroporation (EP) of the injected area. Female mice were transfected with plasmids harboring the enhanced green fluorescent protein (EGFP) gene. Widespread EGFP fluorescence was observed in the mammary epithelial cells of the ducts and adipocytes adjacent to the ducts. As this in vivo gene delivery method is simple, safe, and efficient for gene transfer to the mammary glands, we named this technique “Mammary Intraductal Gene Electroporation” (MIGE). The MIGE method is a useful experimental tool for studies on mammary gland development and differentiation as well as breast cancer research. Full article

Dermal papilla cells (DPCs) serve as the signaling hub regulating hair follicle (HF) development and cyclical growth. This study aims to investigate the biological function and molecular mechanisms of TGFBR1 (transforming growth factor β receptor 1), a differentially expressed gene identified through single-cell transcriptomic sequencing (scRNA-seq) in the DPCs from fine-wool sheep. Primary DPCs were isolated and purified using a combination of enzymatic digestion and mechanical dissociation, followed by immunofluorescence identification (α-SMA and SOX2-positive). Following successful transfection with constructed TGFBR1 overexpression plasmids and siRNA interference vectors, cell proliferation was assessed via EDU staining and CCK-8 assays. mRNA expression of key genes in Wnt/β-catenin, BMP, and Notch signaling pathways (PCNA, CCND1, CTNNB1, SFRP2, BMP2, NOTCH3, SMAD4, etc.) was validated by RT-qPCR. Single-cell transcriptomics revealed significant downregulation of TGFBR1 in DPCs from fine-wool sheep. Functional validation demonstrated that TGFBR1 overexpression markedly suppressed DPC proliferation, whereas knockdown of TGFBR1 expression promoted DPC proliferation. Molecular mechanism studies showed that TGFBR1 overexpression significantly downregulated PCNA, CCND1, CTNNB1, NOTCH3, and SMAD4 while upregulating SFRP2, BMP2, and TGFB1 expression. These findings demonstrate that TGFBR1 acts as a negative regulator of DPCs proliferation by modulating the activity of multiple signaling pathways, including Wnt/β-catenin, BMP, and Notch, thereby suppressing the proliferative capacity of DPCs. This study not only provides new theoretical support for elucidating the role of the TGF-β signaling pathway in H development but also offers theoretical reference for in-depth research on molecular breeding in ultra -fine-wool sheep and the molecular mechanisms underlying HF development. Full article