Search Results (12,102)

Sarcoidosis is a multisystem granulomatous disease of unknown aetiology that frequently presents with mediastinal lymphadenopathy and often requires invasive diagnostic procedures. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is widely used in this setting; however, a definitive diagnosis cannot always be established at first attempt. This study aimed to identify clinical, laboratory, and radiological factors associated with a definitive diagnosis of sarcoidosis in patients referred for EBUS-TBNA. A retrospective analysis was performed including patients undergoing first-time ever EBUS-TBNA for mediastinal lymphadenopathy over a 12-month period. Demographic data, clinical features suggestive of sarcoidosis, chest computed tomography findings, and white blood cell count, were analysed, and definitive diagnoses were established based on cytological results and available follow-up data. Younger age (≤55 years), female sex, the absence of a pulmonary mass >10 mm on imaging, normal white blood cell count, and the presence of clinical features typical of sarcoidosis were significantly associated with a definitive diagnosis of sarcoidosis. Based on these variables, two point-based diagnostic scoring models were developed, demonstrating clinically relevant discriminatory performance. Readily available pre-procedural clinical and radiological factors may assist in estimating the probability of sarcoidosis in patients undergoing EBUS-TBNA for mediastinal lymphadenopathy and may support risk stratification and clinical decision-making. Full article

Soft-tissue sarcomas (STSs) comprise a rare, heterogeneous group of mesenchymal malignancies in which histologic grade remains the strongest determinant of outcome, metastatic risk, and therapeutic strategy. Intermediate/high-grade STSs exhibit a pronounced propensity for early distant relapse, yet growing evidence indicates that metastatic behaviour is not uniform. Within this spectrum, an oligometastatic phenotype, characterised by a limited number of metastases, often confined to the lung, has emerged as a clinically and biologically distinct state associated with more indolent metastatic kinetics and improved survival when treated with aggressive local interventions. However, the criteria that define true oligometastatic STSs remain unsettled, and prospective evidence is lacking. Emerging molecular and immunological correlates provide a potential framework for biological triage. Low genomic complexity (low-risk CINSARC), a B-cell/TLS-rich tumour microenvironment, high immune-cytotoxic signatures, and persistently low or undetectable circulating tumour DNA (ctDNA) are each linked to reduced metastatic competence and may underpin oligometastatic trajectories. Conversely, high chromosomal instability, immunosuppressive microenvironments, and elevated ctDNA levels align with covertly polymetastatic biology despite limited radiographic disease. In this context, artificial intelligence and machinelearning approaches applied to computational genomics, immune profiling, imaging, and liquid-biopsy data offer a powerful strategy to integrate these multi-dimensional features and refine predictions of metastatic behaviour in STS. Oligometastatic STS therefore represents a biologically definable subset amenable to multimodal management integrating local ablative therapies, systemic agents, and immune-based strategies. Prospective, biomarker-stratified trials are needed to validate selection frameworks and optimise treatment sequencing in this evolving therapeutic space. Full article

Over the past two decades, numerous studies have examined the etiological significance of DNA fragmentation in human sperm using methods such as the comet assay (CA), the sperm chromatin structure assay, the sperm chromatin dispersion assay, and the TUNEL assay. We developed single-cell pulsed-field gel electrophoresis techniques, including one-dimensional (1D-SCPFGE) and angle-modulated two-dimensional (2D-SCPFGE), to detect early signs of naturally occurring DNA fragmentation. Comparative studies using purified human sperm with and without DNA fragmentation revealed some technical limitations in the conventional methods. This technical review outlines the procedures to ensure the quantitative performance of SCPFGE: (1) The mass of naked DNA was prepared through simultaneous in-gel swelling and proteolysis, which are highly sensitive to chemical and physical factors. Notably, these processes are vulnerable to reactive oxygen species (ROS). We developed the anti-ROS SCPFGE system to prevent artifactual cleavages. (2) 1D-SCPFGE discharges long-chain fibers from the origin, separating fibrous and granular segments beyond the tips of the fibers. (3) During continuous electrophoresis after 150° rotation (2D-SCPFGE-0-150), long-chain fibers unexpectedly extended diagonally backward from the origin, with long fibrous segments pulled out from a bundle that extended during the first electrophoresis, indicating some fibrous segments were embedded within the long-chain fibers. Even when SCPFGE was employed, one-directional current led to false negatives. (4) 2D-SCPFGE with angle rotation is currently the most sensitive imaging method for single-nuclear DNA fibers. However, without knowing the size of DNA fragments, it remains a semi-quantitative analysis. (5) To prevent artifactual DNA cleavage caused by ice crystals, low-temperature liquid storage is recommended. (6) The in-gel proteolyzed naked DNA is suitable as a substrate for chemical and enzymatic DNA cleavage analyses. Full article

Background: Malignant pleural effusion (MPE) represents a frequent and clinically challenging manifestation of advanced malignancy, particularly in metastatic non-small cell lung cancer (NSCLC). Its management requires integration of diagnostic imaging, symptom-directed therapeutic strategies, and, increasingly, molecular profiling technologies. Recent advancements in this field based on liquid medium (so-called liquid biopsy) have achieved a significant increase in sensitivity, enhancing our ability to investigate biofluids and suggesting their potential integration into standard diagnostic practices, far beyond the canonical plasma biopsies. Fluid obtained from MPE after cytological sample centrifugation is rich in cell-free DNA and less susceptible to nucleic acid degradation during processing, improving overall diagnostic accuracy. Methods: This narrative review summarizes current evidence on the clinical management of malignant pleural effusions in patients with metastatic NSCLC, integrating imaging, procedural management, and molecular profiling from a multidisciplinary tumor board perspective. The primary objective was to synthesize contemporary knowledge with particular attention to the feasibility, reliability, and reproducibility of pleural fluid-based molecular testing. Results: MPE poses diagnostic and therapeutic challenges for all members of the multidisciplinary tumor board, traditionally associated with an adverse prognosis. However, recent advances in cytopathology, histopathology, and liquid-based techniques demonstrate that MPE could be an important source of prognostic or predictive information. At the same time, optimal patient management requires careful integration of imaging findings and procedural strategies (such as pleurodesis or indwelling pleural catheters) with individualized systemic therapy selection. Cell-free DNA in pleural effusions is a promising field of exploration and study, potentially suitable for future guideline implementation, after validation in adequately powered studies, contributing to improving patient management, particularly useful in fragile subsets. Conclusions: The management of MPE in advanced NSCLC is evolving toward a multidisciplinary, precision-oriented model that integrates clinical evaluation, imaging, procedural interventions, and molecular testing. Liquid biopsy technology has gained enough analytical robustness and clinical feasibility to be a useful tool in routine analysis. Biofluid-based molecular testing may have outstanding potential, contributing to improving patient management, avoiding repetitive procedures, and optimizing the overall efficiency and cost-effectiveness of diagnostic practices. Moreover, collaborative projects among different specialties help in consolidating trust in the tumor board decision-making process. Full article

Reactive oxygen species (ROS) are inevitable byproducts of aerobic metabolism that exert a dual role in biological systems. At physiological levels, tightly regulated ROS levels function as essential signaling molecules regulating cellular communication, immune defense, metabolic adaptation, and maintenance of tissue homeostasis. However, excessive or deregulated ROS production disrupts redox balance and contributes to oxidative stress, a key factor in the onset and progression of numerous pathogenesis. This review provides an updated and integrated overview of ROS biology, summarizing their major types, cellular and molecular sources, and physiological functions, highlighting their significance in physiological redox signaling and oxidative stress-mediated disease mechanisms. Key molecular pathways involved in ROS-induced cell damage, redox imbalance, and signaling dysregulation are discussed. In addition, contemporary and emerging approaches for the detection and quantification of ROS and oxidative stress in clinical and preclinical samples—such as biochemical assays, fluorescent probes, biosensors, and advanced imaging techniques—are critically evaluated. The contribution of oxidative stress to the pathophysiology of major disorders, including cancer, diabetes, cardiovascular diseases, neurodegenerative conditions, and inflammatory disorders, is also examined. Finally, this review highlights future perspectives in precision redox medicine, emphasizing the potential of targeted antioxidant-based diagnostic and therapeutic strategies supported by advances in ROS detection technologies and a deeper understanding of redox-regulated biological processes. Full article

Background and Objectives: Male infertility has emerged as a growing global health concern, contributing to 20–30% of all infertility cases. It is a multifactorial condition, arising from genetic, endocrine, structural, environmental and lifestyle factors. This narrative review synthesizes current evidence on epidemiology, diagnostic advances and therapeutic strategies while highlighting emerging trends and research priorities. Materials and Methods: This review adheres to SANRA guidelines. Literature was sourced from PubMed, Saudi Digital Library, Google Scholar, and PsycINFO using MeSH terms including “Male Infertility,” “Diagnosis,” “Treatment,” and “Epidemiology.” Results: Diagnostic evaluation of male infertility includes clinical assessment, advanced semen analysis, imaging techniques, hormonal assays and molecular testing. Despite significant advances in the evaluation of male infertility, idiopathic causes (30–40%) remain challenging. Management strategies include lifestyle modifications, medical therapies including hormones and drugs, surgical interventions, and assisted reproductive technologies (ARTs). However, outcomes remain suboptimal in idiopathic and severe cases, particularly regarding sperm DNA fragmentation and environmental exposures. Conclusions: Substantial knowledge gaps exist in male infertility, particularly in idiopathic cases, molecular mechanisms of environmental pollutants, and long-term ART offspring outcomes. Future research priorities include: (1) molecular and epigenetic biomarkers for improved diagnosis and prognosis; (2) environmental exposure assessment and mitigation strategies; (3) metabolomics-guided personalized therapies; (4) regenerative medicine approaches including spermatogonial stem cell therapy; and (5) multidisciplinary integrative care models. Addressing these gaps through coordinated research and clinical innovation is essential for improving male reproductive health globally. Full article

Background/Objectives: Breast cancer is a heterogeneous and complex disease with significant individual differences in molecular immunophenotype, biological behavior, histopathological morphology, and response to chemotherapy. The presence of tumor-infiltrating lymphocytes (TILs) has gained considerable attention due to growing evidence of their involvement in therapeutic efficacy, particularly in the response to neoadjuvant chemotherapy (NACT). Different immune cell subsets’ frequency, location, and functional orientation vary substantially between tumor types and individuals with apparently identical cancers. Currently, next-generation sequencing (NGS) has provided key insights into the composition of the tumor microenvironment. Simultaneously, immunohistochemistry (IHC) of paraffin-embedded biopsies allows the visualization of marker proteins within the immune infiltrate, thereby enhancing our understanding of the role of immune cells in cancer therapy. Methods: This exploratory study evaluated immune cell tumor infiltration using NGS with immune cell deconvolution, as well as automated IHC on Tru-Cut biopsies from 57 patients with locally advanced breast cancer. Image analysis was performed using Qupath v0.6.0 software. The percentage of infiltrating CD4+ or CD8+ T cells was determined, along with the expression of the markers FoxP3, LAG3, CTLA4, PD1, and TIM-3. We aimed to gain insights into the tumor microenvironment and its influence on the response to NACT in patients with breast cancer. Results: Transcriptomic immune deconvolution approaches suggested that a biased cytotoxic tumor environment is linked to chemosensitivity. IHC assays of individual markers reveal that baseline immune cell abundance and individual checkpoint expression did not differ significantly across the response groups. However, the functional organization and coordination of the tumor immune microenvironment showed distinct associations with chemosensitivity. Conclusions: Features representing immune balance, such as CD8/CD4 ratio and T cell-contextualized metrics, emerged as candidate predictors of pathological response to NACT, outperforming molecular phenotype alone in this exploratory cohort. Full article

There is no approved drug therapy for schwannomas associated with NF2-related schwannomatosis (NF2-SWN). Neither life-saving surgical resection or radiation are curative and can compound the debilitating neurological effects of the schwannomas. We previously identified fimepinostat, a dual histone deacetylase (HDAC)/phosphoinositide-3 kinase (PI3K) inhibitor, as a promising drug candidate with pro-apoptotic effects on NF2-related schwannomas. This preclinical study used the pharmaceutical formulation of fimepinostat to confirm its efficacy in schwannomas and identify pro-apoptotic signaling pathways. Fimepinostat was tested in human schwannoma model cells, patient-derived primary vestibular and non-vestibular schwannoma cells, and in a sciatic nerve allograft model. The signaling pathways leading to caspase-3-dependent apoptosis were elucidated using immune assays, flow cytometry, imaging, proteome, and acetylome analysis. Acute exposure to fimepinostat led to p21-dependent cell cycle inhibition, upregulation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL R2), and downregulation of tumor necrosis factor receptor 1 (TNFR1), Yes-associated protein (YAP), and inhibitors of apoptosis. Moreover, fimepinostat downregulated cytokine and chemokine secretion increased by merlin loss in schwannoma cells. Fimepinostat is a promising new drug intervention for NF2-SWN patients with the potential to promote tumor regression. Full article

Quantitative analysis of leaf cell microstructure is crucial for deciphering agronomic traits in Chinese cabbage, including photosynthetic efficiency, stress tolerance, and yield potential. Traditional manual observation methods are inefficient and highly subjective, failing to meet the demands of large-scale breeding for high-throughput, reproducible microscopic phenotyping. To transition breeding practices from experience-driven to data-driven, there is an urgent need to establish automated, standardized systems for acquiring cell-scale phenotypes. Therefore, this study proposes an automated instance segmentation and phenotyping analysis framework for multi-tissue cells in Chinese cabbage leaf cross-sections. This framework systematically optimizes Mask R-CNN by introducing an attention mechanism to enhance cellular feature responses in complex backgrounds. It employs weighted multi-scale feature fusion to process densely distributed small-scale cells and integrates a refined boundary optimization module to improve recognition accuracy in adherent and blurred regions. On a microscopic image dataset spanning multiple varieties, this method achieves high-precision predictions in instance segmentation tasks. Based on the predicted cell masks, an interactive phenotyping analysis tool was further developed to automatically extract standardized single-cell morphological parameters, including area, perimeter, and Feret’s diameter. The measured parameters exhibit high consistency with manual annotations (correlation coefficients (r) all exceed 0.97). This framework enables high-throughput, standardized phenotypic analysis at the cellular level of leaf cross-sections, providing a reliable method for the digital and automated interpretation of crop microscopic traits. This technical solution not only supports the systematic integration of microscopic phenotypes in Chinese cabbage breeding but also offers a scalable solution for cellular-scale phenotypic research in other crops. Full article

Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of these molecular assemblies remains an enduring biophysical challenge, particularly in cells, where heterogeneity is the rule rather than the exception. Orthogonal signals derived from fluorescence lifetime, fluorescence fluctuations, and fluorescence polarization provide valuable metrics for probing interactions and environments, concentration and size, and rotational dynamics, respectively. This paper combines fluorescence lifetime imaging microscopy with image correlation analysis and polarization to determine the concentrations, brightness, lifetime, and rotational correlation time of different fluorescent states. A two-population model is examined as a prototypical example of a heterogeneous system. The analysis is illustrated on a simple fluorescence model system, where cluster densities, relative brightnesses, lifetimes, and rotational correlation times are extracted. Full article

Background/Objectives: High recurrence rates and intensive lifelong surveillance make bladder cancer among the costliest malignancies to treat. Although Bacillus Calmette–Guérin (BCG) immunotherapy is the standard treatment for high-risk non-muscle-invasive bladder cancer (NMIBC), up to 50% of patients fail to respond, and predictive biomarkers are lacking. Molecular profiling has established three BCG response subtypes (BRS1–3), with BRS3 characterized by an immunosuppressive, BCG-resistant phenotype; however, these features have not been validated at single-cell spatial resolution. Methods: We applied imaging mass cytometry (IMC) to 82 BCG-treated high-risk NMIBC samples and performed (i) single-cell IMC with unsupervised clustering to identify phenotypic cell clusters and quantify cluster abundances and (ii) a convolutional neural network-based gated attention multiple instance learning model trained on IMC images (IMC-GA-MIL) to predict BCG response. Cluster abundances were summarized using II (immune composition within the immune compartment), TT (tumor phenotypic composition), and IT (immune/stromal abundance relative to tumor cells) indices. Results: Single-cell IMC identified 18 distinct phenotypic cell clusters. In BCG responders, immune cells localized within the tumor compartment were enriched and independently protective (HR 0.67, 95% CI 0.49–0.92). BCG nonresponse was associated with a higher abundance of fibroblast-dominant clusters relative to tumor cells (IT index). Plasma cell-dominant clusters were the strongest predictors of progression (II index HR 2.28, 95% CI 1.37–3.79; IT index HR 1.25, 95% CI 1.06–1.48). The IMC-GA-MIL model predicted BCG response with 90% accuracy (9/10) and identified myeloid- and T-cell-associated marker patterns involving CD14, CD11b, CD68, CD8, and FOXP3 as the most informative contributors. Conclusions: Spatial single-cell profiling and IMC-GA-MIL identify spatial immune and stromal features associated with BCG failure. However, findings from both analyses should be considered exploratory and will require validation in larger, independent cohorts. 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

Percutaneous computed tomography (CT) -guided cryoablation is an effective curative treatment for renal cell carcinoma. Improvements in treatment efficacy reflect not only the learning curve but also the integration of multiple adjunctive techniques that can be implemented at different stages of the procedure. Tumour targeting can be enhanced by intravenous contrast administration, or by intra-arterial delivery of contrast medium or iodized oil. Fusion imaging is another option to improve tumour delineation by registering intraprocedural CT with prior cross-sectional imaging. Probe placement for difficult-to-access lesions may be facilitated by alternative access routes, while electromagnetic navigation and robotic systems are being developed as alternatives to manual advancement. To mitigate the cold-sink effect and reduce bleeding risk, transarterial techniques such as embolization or temporary arterial occlusion can be added. Finally, thermoprotective manoeuvres are increasingly used to displace adjacent organs, thereby improving the feasibility, safety, and efficacy of renal cryoablation. Full article

Lymph node metastasis (LNM) is one of the most powerful prognostic determinants in head and neck squamous cell carcinoma (HNSCC). The extent and pattern of nodal involvement critically influence staging accuracy, therapeutic decision-making, and patient outcomes. However, the biological and clinical implications of nodal disease remain complex and continue to evolve. We aim to synthesize current clinical and translational evidence regarding the prognostic and therapeutic impact of LNM in HNSCC and to highlight emerging trends relevant to precision staging. A narrative review was conducted through a structured literature search in PubMed and Scopus (2008–2025), with emphasis on studies published in the last five years. Meta-analyses, large cohort studies, and evidence-based guidelines addressing prognostic factors, biological mechanisms, and management strategies were critically appraised. LNM is consistently associated with reduced overall and disease-free survival across major head and neck subsites. Key independent prognostic variables include the number of metastatic nodes, extranodal extension, and involved cervical levels. Recent advances, such as refinements in the AJCC 8th edition, sentinel lymph node mapping, high-resolution imaging, and molecular profiling, have improved early detection and refined risk stratification. LNM remains central to prognostic evaluation and treatment selection in HNSCC. Integrating biological insights with molecular diagnostics and advanced imaging will be essential to achieving precision staging and individualized therapeutic strategies. Full article

Background/Objectives: Accurate preoperative prediction of cervical lymph node metastasis (LNM) in head and neck squamous cell carcinoma (HNSCC) remains a major clinical challenge. This study aimed to develop a deep learning-based whole-slide image (WSI) model and an integrated nomogram to improve individualized LNM risk stratification. Methods: A total of 355 formalin-fixed paraffin-embedded (FFPE) WSIs and 282 frozen WSIs from the TCGA-HNSC cohort, along with 329 FFPE WSIs from an external institutional cohort, were retrospectively analyzed. Tumor regions were annotated and tiled into standardized patches. A dual-stage multiple instance learning framework was applied to generate WSI-level predictions. A pathological risk score (path-score) was derived and combined with clinical variables to construct a predictive nomogram. Results: The WSI-level model outperformed patch-level classifiers, with the logistic regression-based model achieving area under the curve (AUC) values of 0.821 in the internal validation cohort and 0.730 in the external cohort. The path-score was independently associated with LNM. The integrated nomogram further improved discrimination, yielding AUCs of 0.865 and 0.786 in the internal and external cohorts, respectively. Calibration and decision curve analyses demonstrated good agreement and meaningful clinical benefit. Conclusions: This deep learning-driven pathology nomogram provides a robust and clinically applicable tool for preoperative prediction of cervical lymph node metastasis in HNSCC. Full article