The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment. Significance: This study suggests that Epstein–Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.

Redox imbalance and oxidative stress are increasingly recognized as significant factors in health disorders such as neurodegenerative disorders, premature aging and cancer. However, detecting antioxidant levels that is crucial for managing oxidative stress, can be challenging due to existing assays’ limitations, such as insensitivity to thiol-containing antioxidants. This study presents a simple fluorescence-based assay for antioxidant detection employing the enhanced photocatalytic oxidase-like activity of dithiothreitol (DTT)-assisted bovine serum albumin (BSA)-stabilized gold nanoclusters (DTT@BSA-AuNCs). The reported nanozyme exhibits remarkable stability, versatility, and catalytic activity. Under LED irradiation, DTT@BSA-AuNCs generate singlet oxygen, which converts non-fluorescent thiamine to fluorescent thiochrome, utilizing dissolved oxygen for catalysis. Antioxidants inhibit thiochrome formation, leading to fluorescence quenching. This method enables sensitive detection of antioxidants such as ascorbic acid and glutathione with limits of detection of 0.08 µM and 0.32 µM, respectively, under neutral pH, outperforming previous studies. The assay successfully detects antioxidants in human saliva and cancer cell models. The DTT@BSA-AuNCs-based assay offers a cost-effective, sensitive, and straightforward approach for detecting antioxidants in biological samples, facilitating improved monitoring of oxidative stress in various diseases.

Bimetallic nanoclusters (NCs) hold promise as catalytic materials, yet their potential as enzyme mimics remains largely unexplored. Here, we report the first demonstration that gold–silver nanoclusters (AuAgNCs) function as photo‐responsive oxidase‐like nanozymes. Under visible‐light irradiation, AuAgNCs catalyze thiamine oxidation via a Type I photosensitization pathway, predominantly generating physiologically relevant peroxyl radicals (ROO•). The AuAgNCs outperform monometallic analogs, achieving enhanced catalytic efficiency with lower K _m and higher v _max than previously reported nanozymes. Utilizing these capabilities, we established a rapid (<6 min), reagent‐free platform that enables: (i) selective detection of diverse physiological antioxidants, (ii) quantitative assessment of total antioxidant capacity (TAC) in simulated biofluids and consumer products, and (iii) dynamic tracking of intracellular redox alterations in normal and steatotic hepatocyte models. This versatile platform addresses critical limitations of conventional assays by employing in situ‐generated biologically relevant radicals and high specificity for antioxidant detection, while offering mechanistic insight, operational simplicity, and broad applicability. Our findings expand the functional landscape of bimetallic nanoclusters, establishing them as powerful redox‐active nanozymes for biomedical diagnostics, antioxidant evaluation, and redox biology.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment.Significance:This study suggests that Epstein–Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.