Search Results (10,539)

Background/Objectives: Recombinant adeno-associated virus (rAAV) has become a leading vector in gene therapy. However, manufacturing limitations may result in replication-competent AAV (rcAAV) contamination of clinical rAAV products, posing safety risks. Rigorous testing is therefore essential, and the use of accurately calibrated rcAAV reference standard materials is critical for ensuring assay stability and reliability. A disadvantage of the widely used Tissue Culture Infectious Dose 50 (TCID₅₀) assay is its high variability. This study introduces an optimized TCID₅₀ assay for the precise quantification of infectious rcAAV particles. Methods: We developed a TCID₅₀ assay tailored to rep2-based rcAAV, optimizing key aspects such as viral infection conditions, qPCR reaction systems, and standard curve preparation. We employed an innovative strategy to prepare the standard curve using serial dilutions of rcAAV in cell lysate, ensuring alignment with the test sample matrices. Results: The rcAAV-derived standard curve demonstrated exceptional linearity (R² > 0.99), sensitivity (LOQ ≈ 38 copies), and reproducibility, enabling robust endpoint qPCR analysis. The optimized assay significantly improved the precision of the TCID₅₀ assay, as an inter-assay coefficient of variation (CV) of 11.4% was achieved. Conclusions: This refined TCID₅₀ assay is a reliable method for calibrating infectious titers of rcAAV reference standard materials, thereby enabling the standardization of rcAAV testing. Full article

Epstein–Barr virus (EBV) is a human pathogenic and oncogenic herpesvirus, with worldwide importance, at times associated with serious to life-threatening symptoms, especially in immunocompromised hosts. The available preventive options against EBV disease are limited to medically elaborate and cost-intensive measures of cell-based immunotherapy. The development of novel options of anti-EBV drug targeting is currently a matter of intense international efforts. A putative target of the antiviral therapy approach is the EBV-encoded protein kinase BGLF4, which fulfills a multifaceted role in productive viral replication. So far, viral BGLF4 interactor proteins and phosphorylated substrates have occasionally been reported, but in particular cellular interactors await further characterization concerning both, their relevance for BGLF4 functionality and their accessibility to antiviral drugs. In this study, we have analyzed host cell–BGLF4 interaction, BGLF4 kinase properties, and BGLF4-directed small molecules. The main results are as follows: (i) a mass spectrometry-based interactomic study was performed with EBV-producing Akata-BX1 cells, thereby identifying the human pyruvate dehydrogenase (PDH) as a relevant BGLF4 interactor; (ii) BGLF4–PDH interaction was confirmed by protein coimmunoprecipitation, subcellular cofractionation, and confocal imaging; (iii) the BGLF4-mediated phosphorylation of PDH was demonstrated by an in vitro kinase assay (IVKA); (iv) a reduction in PDH phosphorylation was shown for selected kinase inhibitors, which also exerted BGLF4-directed inhibitory potential in a quantitative qSox-IVKA, and (v) these hit compounds showed anti-EBV activity in lytically induced P3HR-1 cells using qPCR measurement, as well as PDH-inhibitory activity using standardized PDH assays. These data lead to an improved understanding of EBV–host interaction that may open novel anti-EBV preventive opportunities. Combined, the findings point to PDH as a new cellular interactor of the EBV kinase BGLF4. Also, notably, the data on pharmacological intervention with kinase activity or substrate phosphorylation may possibly provide as yet untapped options of antiviral drug targeting. Full article

In glioblastoma, the strong immunosuppression of the tumor immune microenvironment fosters tumor aggressiveness and decreases the effectiveness of therapeutic interventions, including immunotherapies. An intricate network of connections among tumor cells, stroma and infiltrating immune cells sustains immunosuppression. Lectins are immunoregulatory glycan-binding receptors contributing to immunosuppression. Their targeting is proposed as an appealing strategy for anti-cancer therapy. In this work, network-based approaches were exploited to identify a lectin profile that could dissect the complexity of tumor-immunity interactions in glioblastoma. Differential co-expression analysis, employing TCGA, CGGA and GTEx databases (145, 133 and 255 samples, respectively), identified a cluster of novel C-type lectins, with ASGR2 and CLEC12A as principal hubs. Furthermore, TIMER2.0 analysis revealed that their expression was significantly associated with immunosuppressive cells. ASGR2 and CLEC12A expression was also validated by cytofluorimetric analysis on both tumor and liquid biopsies from 20 glioblastoma patients. We report that ASGR2 and CLEC12A C-type lectins are associated with tumor-infiltrating immunosuppressive myeloid subsets and discriminate patients’ poor prognosis. These results suggest that C-type lectins may contribute to the immunosuppressive network sustained by infiltrating myeloid immune cells in GB, resulting in exploitable targets for therapeutic interventions. Full article

Introduction: Stereotactic radiotherapy (SRT) is increasingly used in oligometastatic non-small-cell lung cancer (NSCLC) and is known to elicit systemic immune effects, although the underlying mechanisms remain not fully understood. Methods: In this prospective pilot study, we evaluated plasma cytokine variations in 19 patients with oligometastatic or oligoprogressive NSCLC undergoing SRT. Peripheral blood samples were collected before treatment (T0) and one month after SRT (T1) and the concentrations of nine cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A and TNF-α) were quantified using a multiplex Luminex assay. Non-parametric tests and Cox regression models were used to investigate associations between cytokine levels, clinical variables, systemic treatments, and survival outcomes. SRT induced significant post-treatment increases in IFN-γ, IL-2, and IL-6, consistent with systemic pro-inflammatory activation and T-cell stimulation. Cytokine dynamics were influenced by patient- and tumor-related factors: female sex was associated with higher IL-2 and TNF-α levels; oncogene-addicted tumors showed lower IL-6 levels; and oligoprogressive disease exhibited attenuated cytokine variations compared with metachronous oligometastatic disease. Tyrosine kinase inhibitors were associated with globally reduced cytokine levels and blunted IL-1/IL-2 changes, whereas patients receiving immune checkpoint inhibitors displayed higher IL-2 and IL-6 concentrations and greater post-SRT increases in IFN-γ. Oncogene-addicted status and IL-12 variation emerged as independent predictors of overall survival and a composite model integrating these variables significantly stratified prognosis. Conclusions: These findings suggest that SRT triggers measurable systemic immune activation in oligometastatic NSCLC, which is further shaped by tumor biology, disease burden, and concomitant systemic therapies. Although limited by the small sample size, this study supports the feasibility and potential utility of cytokine profiling to refine patient selection and guide biomarker-driven combinations of SRT with targeted and immune-based treatments, warranting validation in larger prospective cohorts. Full article

Herpes simplex virus (HSV) infections present a major global health burden due to their high morbidity. Conventional therapies offer limited efficacy due to poor bioavailability, the need for frequent administration and potential drug resistance. Recent advances in nanotechnology provide opportunities to overcome these limitations. This review summarizes the latest advances in nanocarrier-based formulations, highlighting their role in improving bioavailability, sustained release, mucosal penetration and antiviral activity. An integrative search was conducted from January 2010 to December 2025. Inclusion and exclusion criteria were used to select the articles. After analyzing the articles, 34 were included in this review with in vitro studies and 14 with in vivo assays. These articles were evaluated in relation to physicochemical characterization studies and in vitro and in vivo assays. Studies were found involving polymeric nanoparticles, metal nanoparticles, solid lipid nanoparticles, liposomes, niosomes, nanoemulsions and nanofibers. Regarding in vitro assays, it was observed that the nanosystems showed increased antiviral activity in cell cultures infected with the herpes simplex virus. In addition, developed nanosystems showed prolonged antiviral activity and lowered toxicity in animal models. Thus, these systems prove to be effective when compared to conventional therapy and can be considered an advance in HSV infection therapy. Full article

Background/Objectives: Mitochondrial Oxidative Phosphorylation (OXPHOS) is a critical metabolic dependency in many cancers. Targeting OXPHOS through Leucine-Rich PPR Motif-Containing Protein (LRPPRC) degrader-mediated OXPHOS Complex Biogenesis Inhibition (OCBI) has demonstrated promising anti-tumor activity. However, rational combination strategies to enhance therapeutic efficacy remain undefined. This study aims to identify FDA-approved drugs that synergize with LRPPRC inhibition and elucidate the underlying mechanism. Methods: We conducted a high-throughput screen of 1376 FDA-approved compounds using LRPPRC isogenic cancer cell models to identify agents that synergize with LRPPRC degrader-based OCBI therapy. The synergistic effects of the candidate compound were validated in multiple cancer cell lines with either genetic ablation or pharmacological inhibition of LRPPRC. Mechanistic studies were performed to investigate the impact on OXPHOS gene expression from both nuclear and mitochondrial genomes. Results: The clinically approved multi-kinase inhibitor Dasatinib was identified as a robust synergistic candidate, exhibiting heightened sensitivity in cancer cells with either LRPPRC knockout or pharmacological inhibition. Mechanistically, Dasatinib selectively suppressed nuclear-encoded OXPHOS genes, whereas LRPPRC inhibition preferentially impaired mitochondrial DNA-encoded OXPHOS genes, resulting in a coordinated dual-genome blockade of OXPHOS. Conclusions: This study uncovers a previously unrecognized synergistic anti-tumor effect between LRPPRC inhibition and Dasatinib, mediated by complementary suppression of nuclear- and mitochondrial genome-encoded OXPHOS pathways. These findings provide a strong mechanistic and translational rationale for combination therapies targeting LRPPRC-high tumors. Full article

Background: The H1N1 influenza A virus evades host immunity through continuous antigenic drift, posing a significant challenge to broad-spectrum neutralizing antibody therapies. This study aims to systematically evaluate the neutralizing capacity of the broad-spectrum antibody C12H5 against H1N1 strains from different eras and identify key immune escape mutation sites. Methods: Three representative H1N1 virus strains from 2009, 2018, and 2023 were selected. An antigen–antibody binding prediction model based on the ESM-2 large language model was constructed by integrating 48,762 GISAID sequence data and deep mutation scanning data from the Bloom laboratory. Candidate escape sites were screened using SHAP (SHapley Additive exPlanations) value analysis. Mutant viruses were constructed via reverse genetics, and their neutralizing capacity and replication fitness were validated through hemagglutination inhibition assays, microneutralization assays, and viral growth kinetics analysis. Results: Machine learning scoring identified five potential escape sites, with K147 exhibiting the highest overall score (0.92). SHAP analysis revealed that the K147 site within the HA protein’s 130-loop region received the highest importance score (0.28), significantly surpassing other candidate sites. Experimental validation revealed that the K147N mutation reduced neutralizing potency against C12H5 by 8-fold (from 1:1024 to 1:128) and approximately 6-fold in microneutralization assays (from 8.3 log₂ to 5.7 log₂), while exhibiting a replication advantage in MDCK cells. Microneutralization assays further confirmed an approximately 6-fold reduction in neutralization sensitivity. Structural analysis indicated that K147 is located at the periphery of the HA receptor-binding domain, immediately adjacent to the receptor-binding site. Conclusions: K147N is identified as the critical mutation mediating C12H5 immune escape, and this mutation has emerged in 2023 circulating strains. This study provides important molecular targets and early warning mechanisms for broad-spectrum antibody optimization and influenza vaccine updates. Full article

The type II cadherin Cadherin-19 (CDH19) plays a crucial role in neural crest development. CDH19 regulates cell–cell junctions and migration by forming catenin–cytoskeleton complexes. Although anti-CDH19 monoclonal antibodies (mAbs) are used for specific applications such as Western blotting and immunohistochemistry (IHC), suitable anti-CDH19 mAbs for flow cytometry are limited. Therefore, developing mAbs that specifically recognize cell-surface-expressed CDH19 is essential for advancing both basic research and therapeutic strategies. Here, novel anti-human CDH19 mAbs (Ca₁₉Mabs) were created using flow cytometry-based high-throughput screening. One clone, Ca₁₉Mab-8 (IgG₁, κ), specifically recognized CDH19-overexpressed Chinese hamster ovary-K1 cells but did not bind to other 21 CDHs (including both type I and type II CDHs) in flow cytometry. Additionally, Ca₁₉Mab-8 recognized endogenous CDH19 in the human glioblastoma cell line LN229. The dissociation constant (K_D) of Ca₁₉Mab-8 for LN229/CDH19 was 9.0 × 10⁻⁹ M. Ca₁₉Mab-8 also detected endogenous CDH19 in Western blotting. Furthermore, Ca₁₉Mab-8 can detect CDH19 in IHC using human melanoma tissue. These findings suggest that Ca₁₉Mab-8 is a novel mAb that detects cell-surface-expressed CDH19 with high specificity and is suitable for various applications in basic research. Therefore, Ca₁₉Mab-8 has potential for clinical diagnosis and tumor therapy. Full article

Repetitive magnetic stimulation (rMS) and static magnetic stimulation (sMS) are currently employed as adjunctive therapies for specific neurological conditions. Despite substantial advances in cancer treatment, unfavorable prognoses and outcomes persist, especially for aggressive tumors, including glioblastoma and acute myeloid leukemia. The utilization of magnetic fields has shown antitumoral benefits in both in vitro and animal studies, suggesting its potential as an efficient non-invasive therapeutic approach; nevertheless, the precise mechanisms of action remain unclear. This scoping review intended to identify published research investigating the effects of sMS and rMS in in vitro and in vivo models to evaluate their impacts on morphological and molecular parameters. Four databases (PubMed, Embase, Web of Science, and Scopus) were assessed; the search strategy was limited to the past twenty-five years of data publication. Studies utilizing rMS or sMS as a treatment for conditions other than cancers, as well as those not considering these therapies as adjunctive therapy, were eliminated. Nine articles using rMS were included: three in vitro, two employing animal models, and the remaining four including both cellular and animal-based analyses. Sixteen studies using sMS were identified: twelve in vitro, three in vivo, and one with both models. The findings show that both rMS and sMS elicit a diverse array of biological responses in cancer cells, which are very variable and greatly influenced by tumor type, stimulation frequency, magnetic field intensity, exposure length, and experimental conditions. Full article

Background: Preterm birth is a leading cause of neonatal mortality and long-term disability worldwide. Injury in premature infants is demonstrated by disrupted organ development from inflammation, oxidative stress, hypoxia, and impaired vascular maturation. Current therapies largely provide supportive care and do not directly promote tissue regeneration. Mesenchymal stem cell (MSC)-based therapies have emerged as a potential strategy to enhance endogenous repair across organ systems commonly affected by prematurity. Results: Evidence indicates that MSCs exert therapeutic effects primarily through transient paracrine signaling rather than long-term engraftment. Following administration, MSCs release cytokines, growth factors, and extracellular vesicles that reduce inflammation, promote angiogenesis, and support tissue repair. In preclinical models of neonatal brain injury, MSC therapy has been associated with improved oligodendrocyte maturation and reduced white matter injury. Early clinical trials in neonatal encephalopathy demonstrate feasibility and short-term safety of both autologous and allogeneic cell products. However, studies remain limited by small sample sizes and short follow-up. Cell-free approaches using MSC-derived extracellular vesicles may offer similar biological benefits with potentially lower safety and regulatory concerns. Conclusions: MSC-based therapies represent a promising regenerative approach for complications of prematurity. Rigorous, large-scale trials with standardized protocols and long-term follow-up are necessary to clarify efficacy, optimize delivery strategies, and define safety in this vulnerable population. Full article

Background/Objectives: Research on the association between change from baseline in patient-reported outcomes (PROs) and overall survival (OS) in patients with advanced non-small-cell lung cancer (NSCLC) exists. This study evaluated the association between post-baseline PROs and OS in patients with advanced NSCLC who received first-line cemiplimab-based therapy. Methods: We evaluated PRO data from two phase III studies (EMPOWER-Lung 1 [NCT03088540] and EMPOWER-Lung 3 [NCT03409614]) using a Cox proportional hazards model. Twelve pre-specified PRO scales from the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 and Quality of Life Questionnaire Lung Cancer 13 module were evaluated. Landmark analyses were conducted at 3, 6, 9, and 12 months. Time-dependent analyses using change from baseline PROs as a time-dependent covariate were conducted to evaluate the association of post-baseline PRO improvement with OS. Results: At the 3-month landmark, we observed a 56% reduction in the risk of death (HR = 0.44; 95% CI: 0.32–0.62; nominal p < 0.0001) among stable/improved vs. worsened/unobserved PROs for global health status (GHS)/quality of life (QoL). Results at the 6-, 9-, and 12-month landmarks of GHS/QoL were consistent with those at the 3-month landmark. Time-dependent analyses showed that every 10-point improvement in GHS/QoL was associated with a 31% reduction in the risk of death (HR = 0.69; 95% CI: 0.64–0.75; nominal p < 0.0001). Conclusions: In patients with advanced NSCLC who received first-line cemiplimab-based therapy, improvements in post-baseline PROs are associated with improved OS. These results may inform endpoint selection and interpretation of future clinical trials. Full article

Wilms tumor (WT) is the most common malignant renal tumor in childhood and represents one of the major success stories of pediatric oncology, with very good survival achieved through risk-adapted multimodal therapy. Nevertheless, a subset of patients—particularly those with diffuse anaplasia, blastemal-type tumors persisting after chemotherapy, or relapsed disease—continues to experience poor outcomes and significant long-term treatment-related morbidity. These challenges highlight the need for novel therapeutic strategies beyond conventional cytotoxic approaches. Growing evidence indicates that WT is characterized by a complex and distinctive tumor microenvironment (TME) shaped by its developmental origin and triphasic histology. Immune cell infiltration, inflammatory mediators, and immune checkpoint pathways interact differently with blastemal, epithelial, and stromal tumor components, generating heterogeneous immune surveillance and escape mechanisms. In particular, tumor-associated macrophages (TAMs), functionally impaired natural killer (NK) cells, and immunosuppressive stromal elements play a central role in shaping an immune milieu that may limit the efficacy of immune-based therapies. Although immunotherapy has changed the management of several adult malignancies and some pediatric cancers, its translation to WT has so far been limited, with modest results in unselected patient populations. Recent immunogenomic and proteogenomic studies, however, suggest the existence of biologically distinct WT subsets with different immune features and potential susceptibility to targeted immunotherapeutic approaches. This narrative review integrates pathological, immunological, and clinical perspectives to summarize current knowledge on the WT immune microenvironment, mechanisms of tumor immune evasion, and emerging immunotherapeutic strategies. By providing a unified framework, it aims at supporting a multidisciplinary approach for the rational development of future immune-based and combination therapies tailored to specific WT subgroups. Full article

Background/Objectives: Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and remains associated with poor prognosis despite multimodal therapy. Metabolic reprogramming, particularly increased dependence on glutamine, supports GBM bioenergetic, biosynthetic, and redox demands. This study aimed to systematically identify glutamine-associated metabolic regulators with prognostic relevance and biological plausibility in GBM. Methods: Transcriptomic data from TCGA and GTEx were analyzed using GEPIA2, with survival validation performed using the CGGA. Functional pathway enrichment, protein expression assessment, protein–protein interaction network analysis, tumor microenvironment evaluation, epigenetic profiling, and single-cell RNA sequencing validation were integrated to contextualize candidate genes. Pharmacogenomic correlation analysis and structure-based molecular docking were applied as supportive validation layers. Results: Ceruloplasmin (CP), Solute Carrier Family 25 Member 13 (SLC25A13), and Solute Carrier Family 38 Member 2 (SLC38A2) were selectively dysregulated and associated with poor clinical outcomes in GBM. CP was linked to redox regulation and stress-adaptive survival programs, SLC25A13 to mitochondrial metabolite exchange and glutamine-coupled nucleotide biosynthesis, and SLC38A2 to glutamine uptake, nutrient sensing, and mTORC1-MYC-associated growth signaling. Conclusions: CP, SLC25A13, and SLC38A2 emerge as clinically relevant glutamine-associated metabolic regulators in GBM, linking redox regulation, mitochondrial metabolite exchange, and glutamine-driven growth signaling. These findings highlight transport- and exchange-centered metabolic nodes as potential biomarkers and candidates for future metabolic targeting in GBM. Full article

Bloodstream infection (BSI) is a serious clinical condition associated with high morbidity and mortality, requiring rapid identification of causative agents and timely antimicrobial susceptibility testing (AST). This study evaluated accelerated bacterial identification from positive blood culture samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with two rapid processing approaches: a serum separation tube-based centrifugation method (SST method) and shortened cultivation on solid media. Rapid identification was followed by accelerated AST, performed either from a bacterial cell pellet (SST method) and from early-grown bacterial biomass (shortened cultivation protocol). The results were compared with those obtained using routine laboratory procedures. A total of 270 positive blood culture samples were analyzed, with 135 samples processed by each protocol. Both approaches achieved an identification success rate of 93.33%. Rapid AST using the SST method showed error rates of 0.51% minor errors, 0.57% major errors, and 0.23% very major errors, with an overall agreement of 98.69%. The shortened cultivation protocol demonstrated lower error rates (0.46% minor errors and 0.23% major errors) and an overall agreement of 99.31%. These findings confirm that MALDI-TOF MS enables reliable early identification of BSI pathogens and rapid AST, supporting timely optimization of antimicrobial therapy and early detection of multidrug-resistant strains. Full article

Background/Objectives: Despite recent advances in breast cancer diagnostics, therapies and personalized medicine through genetic profiling, effective treatment of aggressive subtypes, particularly triple-negative breast cancer (TNBC), remains a considerable clinical challenge. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that influence tumor progression and are detectable extracellularly in biofluids, where they are typically protected within extracellular vesicles (e.g., exosomes) or associated with RNA-binding proteins and lipoprotein complexes. This review integrates current evidence on oncogenic and tumor-suppressive extracellular miRNAs in breast cancer, with emphasis on subtype-specific functions and potential clinical relevance as liquid-biopsy biomarkers and therapeutic targets. Methods: A PubMed-based literature review (January 2000–February 2026) was conducted using search terms combining “breast cancer” with “miRNA/microRNA” and “circulating/plasma/serum/exosomal/extracellular vesicle.” Studies were prioritized if they provided validated targets/mechanisms and/or human clinical evidence for diagnostic, prognostic, or predictive utility; discrepant findings were evaluated in a subtype-aware framework. Findings were organized into functional categories (e.g., EMT/metastasis, cell-cycle/DNA damage, immune modulation, and hormone/growth factor signaling). Clinical and translational studies evaluating circulating miRNAs for diagnosis, prognosis, treatment response, and toxicity prediction were synthesized, together with key pre-analytical and analytical variables that affect reproducibility. Results: Across mechanistic and clinical studies, miR-21 and miR-155 recur as prominent oncogenic miRNAs, whereas miR-205 is frequently reported as a tumor-suppressive miRNA that is reduced in breast cancer and in circulation in several cohorts. Panels combining these miRNAs show promise for sensitive and specific breast cancer diagnostics. Additionally, several miRNAs show context- or subtype-dependent effects, with opposing activities reported between TNBC and estrogen receptor (ER)-positive disease (e.g., miR-17-92, miR-425, miR-181 family members, miR-31, and miR-24). Conclusions: Circulating miRNAs represent a promising class of minimally invasive biomarkers and potential therapeutic targets; however, translation is constrained by biological context dependence and by pre-analytical and analytical variability. Standardized protocols and rigorously validated, subtype-aware biomarker panels will be essential for clinical implementation and for enabling miRNA-informed precision oncology in breast cancer. Full article