Search Results (1,054)

Background/Objectives: The emergence of immune-evasive SARS-CoV-2 variants highlights the need for adaptable vaccine strategies. Trimeric receptor-binding domain (tRBD) antigens offer structural and immunological advantages over monomeric RBDs, but DNA vaccine efficacy has been limited by inefficient antigen expression, particularly in non-dividing antigen-presenting cells. Although cytoplasmic transcription–based DNA platforms have been developed to overcome nuclear entry barriers, their utility for antigen structure–function optimization remains underexplored. This study evaluated whether integrating a rationally designed trimeric RBD with a T7-driven cytoplasmic transcription system could enhance immunogenic performance. Methods: A DNA vaccine encoding a tandem trimeric SARS-CoV-2 RBD was delivered using a T7 RNA polymerase-driven cytoplasmic transcription system. In vitro antigen expression was assessed following Lipofectamine 3000-mediated transfection. In vivo, mice were immunized with the SM-102-based Rpol/tRBD/LNP formulation, and immunogenicity was assessed by antigen-specific antibody titers, serum neutralizing activity, and T-cell response profiling, together with basic safety/tolerability evaluations. Results: The T7-driven cytoplasmic transcription system markedly increased antigen mRNA and protein expression compared with conventional plasmid delivery. Rpol/tRBD vaccination induced higher anti-RBD IgG titers, enhanced neutralizing antibody activity, and robust CD8⁺ T cell responses relative to monomeric RBD and plasmid-based trimeric RBD vaccines. Immune responses were Th1-skewed and accompanied by germinal center activation without excessive inflammatory cytokine induction, body-weight loss, or hepatic and renal toxicity. Conclusions: This study demonstrates that integrating rational trimeric antigen engineering with direct cytoplasmic transcription enables balanced and well-tolerated immune activation in a DNA vaccine context. The T7 autogene-based platform provides a flexible framework for antigen structure–function optimization and supports the development of next-generation DNA vaccines targeting rapidly evolving viral pathogens. Full article

The flavivirus NS1 protein is a component of the viral replication complex and plays diverse, yet poorly understood, roles in the viral life cycle. To enable real-time visualization of the developing replication organelle and biochemical analysis of tagged NS1 and its interacting partners, we engineered a replication-competent yellow fever virus (YFV) replicon encoding a C-terminal fusion of NS1 with green fluorescent protein (NS1–GFP). The initial variant was non-viable in the absence of trans-complementation with wild-type NS1; however, viability was partially restored through the introduction of co-adaptive mutations in GFP (Q204R/A206V) and NS4A (M108L). Subsequent cell culture adaptation generated a 17-nucleotide frameshift within the NS1–GFP linker, resulting in a more flexible and less hydrophobic linker sequence. The optimized genome, in the form of a replicon, replicates in packaging cells that produce YFV structural proteins, as well as in naive BHK-21 cells. In the packaging cells, the adapted NS1–GFP replicon produces titers of infectious particles of approximately 10^6 FFU/mL and is genetically stable over five passages. The expressed NS1–GFP fusion protein localizes to the endoplasmic reticulum and co-fractionates with detergent-resistant heavy membranes, a hallmark of flavivirus replication organelles. This NS1–GFP replicon provides a novel platform for studying NS1 functions and can be further adapted for proximity-labeling strategies aimed at identifying the still-unknown protease responsible for NS1–NS2A cleavage. Full article

Non-AIDS-defining cancers (NADCs) have emerged as an increasingly prominent cause of non-AIDS-related morbidity and mortality among people living with HIV (PLWH). However, the scarcity of NADC clinical samples, compounded by privacy and security constraints, continues to present formidable obstacles to advancing pathological and clinical investigations. In this study, we adopted a joint analysis strategy and deeply integrated and analyzed transcriptomic data from 12,486 PLWH and cancer patients to systematically identify potential key regulators for 23 NADCs. This effort culminated in NADCdb—a database specifically engineered for NADC pathological exploration, structured around three mechanistic frameworks rooted in the interplay of immunosuppression, chronic inflammation, carcinogenic viral infections, and HIV-derived oncogenic pathways. The “rNADC” module performed risk assessment by prioritizing genes with aberrant expression trajectories, deploying bidirectional stepwise regression coupled with logistic modeling to stratify the risks for 21 NADCs. The “dNADC” module, synergized patients’ dysregulated genes with their regulatory networks, using Random Forest (RF) and Conditional Inference Trees (CITs) to identify pathogenic drivers of NADCs, with an accuracy exceeding 75% (in the external validation cohort, the prediction accuracy of the HIV-associated clear cell renal cell carcinoma model exceeded 90%). Meanwhile, “iPredict” identified 1905 key immune biomarkers for 16 NADCs based on the distinct immune statuses of patients. Importantly, we conducted multi-dimensional profiling of these key determinants, including in-depth functional annotations, phenotype correlations, protein–protein interaction (PPI) networks, TF-miRNA-target regulatory networks, and drug prediction, to deeply dissect their mechanistic roles in NADC pathogenesis. In summary, NADCdb serves as a novel, centralized resource that integrates data and provides analytical frameworks, offering fresh perspectives and a valuable platform for the scientific exploration of NADCs. Full article

Porcine epidemic diarrhea virus (PEDV) has emerged as a major pathogen responsible for porcine diarrheal diseases, causing outbreaks of severe diarrhea and high mortality in neonatal piglets, thereby inflicting severe economic losses on the global swine industry. Current commercial PED vaccines, comprising conventional inactivated and live attenuated formulations, have exhibited progressively diminished efficacy in the face of emerging PEDV variants. The development of high-efficiency vaccine platforms is therefore critical for PED control. This study engineered a cellular membrane nanovesicle (CMN)-based vaccine, which differs from existing inactivated or subunit vaccines by presenting the PEDV spike (S) protein on the cell membranes to mimic the bilayer phospholipid structure of the viral envelope. The full-length S protein (FS, aa 19-1309) or a truncated S protein fragment (TS, aa 19-726) was expressed in Expi293F cells, followed by extraction of cell membranes to assemble antigen-displaying CMN vaccines. Compared with commercial live attenuated vaccine, administration of the CMN vaccine elicited high-titer neutralizing antibodies and elevated IFN-γ-producing CD8⁺ T cells in murine studies. Safety assessments revealed no adverse effects on body weight, hepatic/renal function indices, or histopathological parameters in vaccinated mice. Furthermore, immunization of piglets elicited notable humoral and CD8⁺ T cell immune responses. Collectively, the strategy of CMN-based vaccine described herein delivers a potential PEDV vaccine platform, thereby offering a novel avenue for next-generation veterinary vaccine development. Full article

Background: Self-amplifying mRNA (sa-mRNA) represents a promising platform for vaccines and gene therapies, offering sustained protein expression at low doses through self-replication. For vaccines targeting respiratory pathogens, pulmonary delivery of sa-mRNA lipid nanoparticles (LNPs) is particularly advantageous, enabling direct delivery to the infection site and induction of mucosal immunity. Objective: In this study, we evaluated the stability of sa-mRNA–LNPs under refrigerated and frozen conditions and developed a dry powder formulation suitable for inhalation, produced by freeze-drying followed by cryomilling with leucine. Methods: sa-mRNA–LNPs formulated in HEPES buffer with 20% (w/v) sucrose were stored for up to 8 weeks as liquid or freeze-dried samples at various temperatures (−80 °C, −20 °C, 4 °C, and 20 °C). Biological stability was assessed by transfection efficiency in HeLa cells, while physical stability was characterized by encapsulation efficiency, zeta potential, particle size, and polydispersity index. Results: Liquid formulations remained stable for at least 8 weeks at −80 °C and −20 °C but rapidly lost stability at 4 °C and 20 °C. Freeze-drying effectively preserved sa-mRNA–LNP functionality and structural integrity for up to 8 weeks at 4 °C, with only minor structural changes. Subsequent cryomilling in the presence of 4 wt-% leucine produced a respirable dry powder while retaining approximately 60% of the original sa-mRNA–LNP functionality. Although cryomilling induced some structural alterations, the remaining functional fraction remained stable during storage. The resulting powders displayed favorable aerosol performance for deep lung delivery, as demonstrated by cascade impaction (MMAD = 4.13 ± 0.26 µm). Conclusions: In conclusion, freeze-drying effectively preserved sa-mRNA–LNP integrity at 4 °C, whereas cryomilling with leucine produced a respirable dry powder suitable for pulmonary delivery, providing a foundation for globally accessible, needle-free sa-mRNA vaccines against respiratory diseases. Full article

Rabies is a neglected tropical zoonotic disease caused by rabies-virus (RV) infection and is responsible for almost 60,000 annual deaths globally, largely affecting the socio-economically disadvantaged population. Although fatality is preventable by immunization either before or after exposure with therapeutic antibodies, the high cost of prophylaxis or treatment limits their accessibility for the affected population. However, due to the almost 100% fatality rate in symptomatic individuals, almost 29 million annual vaccinations are performed, imposing high financial burden. Human transmission occurs principally through bites from infected dogs and although multiple mammalian species are permissive to RV, transmission from them or from symptomatic humans is rare. To overcome the limitations posed by the requirement of biosafety level-3 (BSL-3) containment for live virus culture, we established a replication-deficient vesicular stomatitis virus (VSV) pseudovirus expressing the Rabies-G (RV-G) protein and a multiplexed Luminex immunoassay for quantifying anti-rabies antibodies in equine sera. The purified pseudovirus exhibited robust luciferase activity and was able to infect multiple mammalian cell lines, although with variable efficiency. Using hyper-immunized equine serum, we observed a strong correlation (ρ > 0.9, p < 0.001) between binding antibody titers measured by the Luminex assay with neutralizing antibody titers determined using the pseudovirus-based neutralization assay. These assays provide a safe, quantitative, and BSL-2-compatible platform for rabies serological evaluation and vaccine testing. Full article

Background/Objectives: Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer (NSCLC), is frequently diagnosed at advanced stages with distant metastasis, underscoring the need for effective prognostic biomarkers. Fibroblast growth factor 2 (FGF2), a multifunctional regulator, has shown to play contradictory roles in cancer progression. Methods: We analyzed three independent Gene Expression Omnibus (GEO) datasets (GSE19804, GSE18842, and GSE19188) to identify consistently dysregulated genes in LUAD. Functional enrichment (GO, KEGG, and cancer hallmark analysis), protein–protein interaction (PPI) network construction, and hub gene prioritization were performed using public bioinformatic tools. Survival analyses were conducted via the Kaplan–Meier Plotter. The expression of FGF2 was validated across multiple platforms, including TCGA, CPTAC, TNMplot, LCE, and the Human Protein Atlas. Functional assays (Transwell migration and wound healing) demonstrated that exogenous FGF2 significantly suppressed LUAD cell motility in vitro. Results: A total of 949 differentially expressed genes (DEGs) were commonly identified across datasets, with enrichment in cell adhesion and metastasis-related pathways. Among the 11 hub genes identified, FGF2 was consistently downregulated in LUAD tissues across all datasets and stages. Higher FGF2 expression was associated with longer overall and progression-free survival. In vitro, FGF2 treatment significantly suppressed the migration and wound healing abilities of LUAD cell lines. Conclusions: FGF2 is downregulated in LUAD and inversely associated with metastatic progression and poor prognosis. The observed reduction in cancer cell motility upon FGF2 treatment in vitro, together with its expression pattern, supports a potential tumor-suppressive role and suggests that FGF2 may serve as a candidate non-invasive biomarker for monitoring LUAD metastasis. Full article

Background: Diabetic nephropathy (DN) is largely driven by transforming growth factor-β1 (TGF-β1)-mediated fibrosis. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes offer precise gene disruption, yet effective non-viral delivery remains a challenge. This study developed cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for intracellular delivery of TGFB1-targeting RNP as an early-stage platform for DN gene modulation. Methods: A single-guide RNA (sgRNA) targeting human TGFB1 was assembled with Cas9 protein (1:1 and 1:2 molar ratios). Hybrid NLCs comprising squalene, glyceryl trimyristate, and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were formulated via optimized emulsification–sonication to achieve sub-100 nm particles. Physicochemical properties, including polydispersity index (PDI), were assessed via dynamic light scattering (DLS), while silencing efficacy in HEK293T cells was quantified using quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Results: Optimized NLCs achieved hydrodynamic diameters of 65–99 nm (PDI < 0.5) with successful RNP complexation. The 1:2 Cas9:sgRNA formulation produced the strongest gene-editing response, reducing TGFB1 mRNA by 67% (p < 0.01) compared with 39% for the 1:1 ratio. This translated to a significant reduction in TGF-β1 protein (p < 0.05) within 24 h. Conclusions: DOTAP-based hybrid NLCs enable efficient delivery of CRISPR–Cas9 RNP and achieve significant suppression of TGFB1 expression at both transcriptional and protein levels. These findings establish a promising non-viral platform for upstream modulation of profibrotic signaling in DN and support further evaluation in kidney-derived cells and in vivo renal models. Full article

Severe acute respiratory syndrome coronavirus (SARS-CoV) assembles and buds from the Golgi apparatus or the ER membrane, but the specific membrane microdomains utilized during this process remain underexplored. Here, we show that co-expression of the SARS-CoV structural proteins S, M, and N in HEK-293T cells is sufficient to generate genome-free SARS-CoV-like virus-like particles (VLPs), which preferentially bud from glycolipid-enriched membrane lipid raft microdomains. Immunofluorescence microscopy using raft-selective dyes (DiIC16) and spike-specific antibodies revealed strong co-localization of VLPs with lipid rafts. Detergent-resistant membrane analysis and sucrose gradient centrifugation further confirmed the presence of S protein in buoyant, raft-associated fractions alongside the raft marker CD44. Importantly, pharmacological disruption of rafts with methyl-β-cyclodextrin reduced VLP budding and S protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. Additionally, our data support lipid raft-associated proteins’ (e.g., FNRA, VIM, CD59, RHOA) roles in modulating cellular responses conducive to viral replication and assembly. These findings highlight lipid rafts as crucial platforms for SARS-CoV morphogenesis and suggest new avenues for vaccine and antiviral development using VLPs and raft-targeting therapeutics. 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

Breast cancer is the leading cause of cancer-related mortality in African American (AA) women. In this study we evaluated the serum proteomic profile of AA women with breast cancer using an integrated proteomic framework with multivariate pattern analysis. Using 2D-DIGE, thousands of serum protein spots were detected across 33 gels; 46 spots met criteria for presence, statistical significance, and differential expression. Proteins from the spots were identified by MALDI-TOF/TOF and matched in curated databases, highlighting serum biomarkers including ceruloplasmin, alpha-2-macroglobulin, complement component C3 and C6, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein and haptoglobin-related protein. LC–MS/MS analysis revealed 163 differentiating peptides after imputing and filtering 286 peptides. These were evaluated using cumulative distribution function (CDF) analysis, a nonparametric method suited for limited sample sizes. Peptide patterns were explored with Random Forest, showing concordance with CDF. The model achieved an AUC of 0.85 at the peptide level. This workflow identified differentiating proteins (CERU, A2MG, CO3, VTDB, HEMO, APOB, APOA4, CFAH, CO4A, AACT, K1C10, ITIH2, ITIH4), highlighting CERU, A2MG, and CO3 with overexpression and reproducible identification across platforms. We present an integrated, non-invasive serum protein biomarker signature panel specific to AA women, through reproducible proteomic sensor framework to support early detection and breast cancer prevention. Full article

Liver fibrosis is characterized by an excessive accumulation of extracellular matrix (ECM) proteins as a wound-healing response to chronic liver injury, leading to tissue scarring and organ dysfunction. Natural compounds, including phytonutrients and polyphenols, have been shown to exert protective effects by reducing profibrotic biomarkers in vitro and in vivo models. Here, we provide the first evidence that the polyphenol hesperidin (HE) can counteract the onset of fibrotic responses in an ex vivo mouse liver fibrosis model induced by Transforming Growth Factor-β1 (TGF-β1) (5 ng/mL). Notably, HE drives early ECM remodeling in the fibrotic mouse liver tissue. Fibrosis-related parameters were assessed at both the transcriptional and translational levels after treatment with HE at increasing concentrations of 50, 75, and 100 µg/mL. Interestingly, HE at 75 µg/mL exerted the strongest beneficial effect, significantly decreasing the gene expression of α-SMA, SERPINH-1, FN-1, VIM and COL1A1 and counteracting the TGF-β1-induced upregulation of key fibrotic markers, including α-SMA, COL1A2, and VIM, reflecting its capacity to attenuate myofibroblast activation and ECM production and modulating membrane lipid peroxidation. Furthermore, HE inhibited SMAD2 phosphorylation, suggesting that its antifibrotic activity may involve the modulation of the TGF-β/SMAD signaling pathway. Moreover, it promoted an anti-inflammatory response, due to a decrease in IL-1β and IL-6 expression. Our study highlights the potential of the ex vivo model as a platform for evaluating the antifibrotic efficacy of natural molecules, and it suggests significant translational implications and new opportunities for developing innovative therapeutic strategies. Full article

Heterogeneous expression of a single surface protein within one cell population can drive major functional differences, yet low-expression subtypes remain difficult to isolate. Conventional tube-based immunomagnetic separation collapses all labelled cells into one positive fraction and thus cannot resolve small differences in marker abundance. Here, we present MagSculptor, a microfluidic platform for high-resolution magnetic fractionation of low-expression EpCAM-defined subtypes within one immunomagnetically labelled population at a time. Arrays of soft-magnetic strips create localized high-gradient zones that map modest differences in bead loading onto distinct capture positions, yielding High (H), Medium (M), Low (L), and Negative (N) fractions. Finite element method simulations of coupled magnetic and hydrodynamic fields quantify the field gradients and define an operating window. Experimentally, epithelial cancer cell lines processed sequentially under identical settings show reproducible subtype partitioning. In a low-EpCAM model (MDA-MB-231), conventional flow cytometry, under standard EpCAM staining conditions, did not yield a robust EpCAM-positive gate, whereas MagSculptor still revealed graded subpopulations. Western blotting confirms a monotonic decrease in EpCAM abundance from H to N, and doxorubicin assays show distinct in vitro drug sensitivities, while viability remains above 95%. MagSculptor thus helps extend immunomagnetic separation from binary enrichment to multi-level isolation of low-expression subtypes and provides a convenient front-end for downstream functional and molecular analyses. Full article

Non-coding RNAs have emerged as central regulators of gene expression in neurodegenerative diseases, offering new opportunities for diagnosis and therapy. This review synthesizes current knowledge on microRNAs, long non-coding RNAs, and circular RNAs in Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, emphasizing their roles in synaptic function, proteostasis, mitochondrial biology, and neuroinflammation. We evaluate evidence supporting non-coding RNAs as circulating and tissue-based biomarkers for early detection, disease monitoring, and patient stratification, and we compare analytical platforms and biofluid sources. Mechanistic insights reveal how non-coding RNAs modulate pathogenic protein aggregation, neuronal excitability, immune cell crosstalk, and blood–brain barrier integrity. Translational efforts toward RNA-targeted interventions are reviewed, including antisense oligonucleotides, small interfering RNAs, miRNA mimics and inhibitors, circular RNA decoys, and extracellular vesicle-mediated delivery systems. We discuss pharmacological modulation, delivery challenges, safety concerns, and strategies to enhance specificity and CNS penetration. Finally, we outline emerging computational and multi-omics approaches to prioritize therapeutic targets and propose a roadmap for advancing non-coding RNA research from preclinical models to clinical trials. Addressing biological heterogeneity and delivery barriers will be pivotal to realizing the diagnostic and therapeutic promise of the non-coding transcriptome in neurodegenerative disease. Collaboration across disciplines and rigorous clinical validation are urgently needed. Full article