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PubMed2026

Protocol for the isolation and characterization of extracellular vesicles and particles from human and murine cell lines.

Cells produce a heterogeneous population of extracellular vesicles and particles (EVPs). Small extracellular vesicles (sEVs or exosomes) are lipid membrane-enclosed vesicles with sizes ranging from 30 to 150 nm. Here, we present a protocol to isolate and characterize EVPs from the conditioned medium of cell lines. We describe steps for cell culture, conditioned media collection, differential ultracentrifugation, and EVP characterization. We then detail procedures for proteomic and biodistribution analyses. For complete details on the use and execution of this protocol, please refer to Yeung et al.1.

Animals↗

PubMed2025

Identification of molecular markers and exploration of the oncogenic role of exomeres in hepatocellular carcinoma.

Extracellular vesicles and particles (EVPs) serve as functional mediators delivering their cargoes to specific destinations. Exomeres (EMs) represent a newly discovered subset of nanoparticles, with limited understanding of their biophysical characteristics and functionalities. Here, we isolated and studied EMs from different normal and cancer cell lines. Proteomic analysis reveals distinctive features of EMs compared to small extracellular vesicles (sEVs) and identifies galactosamine (N-acetyl)-6-sulfatase (GALNS) and mannosidase alpha class 2B member 1 (MAN2B1) to be expressed in EMs, indicating their potential as specific EM molecular markers. Subsequent investigations into tumor-derived EMs demonstrate their oncogenic properties to support cancer growth and metastasis. Furthermore, analysis of murine hepatocellular carcinoma-derived EM reveals their ability to induce cell cycle progression and metabolic alterations. Collectively, our data highlight the distinct nature of EMs as a nanoparticle subpopulation different from sEVs, with cancer-derived EMs significantly contributing to tumor growth and dissemination.

Carcinoma, Hepatocellular↗

PubMed2026

Host metadherin coordinates hepatic lipid metabolism and CD8+ T cell immunity to promote tumor progression.

Cancer progression is systemically influenced by distant organ dysfunction induced by primary tumors, yet how long-distance tumor-organ crosstalk regulates antitumor immunity remains unclear. Here, we identify host metadherin (MTDH) as a critical regulator of tumor-induced immunosuppression and metabolic reprogramming via tumor-liver interactions. Using Mtdh knockout mouse models, we show that concurrent MTDH loss in hepatocytes and CD8+ T cells enhances effector T cell function and suppresses tumor growth and metastasis. Mechanistically, tumor-derived extracellular vesicles and particles (EVPs) activate Kupffer cells to secrete tumor necrosis factor α (TNF-α) and TGF-β, which suppress hepatic PPARα-mediated lipid oxidation via nuclear factor κB (NF-κB) signaling. MTDH loss restores hepatic lipid catabolism, reduces systemic lipid levels, and promotes mitochondrial metabolic reprogramming in CD8+ T cells under lipid-reduced conditions, thereby boosting antitumor immunity. Genetic or pharmacological targeting of MTDH synergizes with anti-PD-1 therapy. These findings establish host MTDH as a key mediator of tumor-liver crosstalk through metabolic and immune interactions, driving systemic cancer progression.

CD8(+) T cells↗

PubMed1979

Application of electron probe X-ray microanalysis to calcification studies of bone and cartilage.

The use of electron probe x-ray microanalysis in previous studies of bone and cartilage has been reviewed with emphasis on the results which have contributed to some of the current concepts of the mechanism of mineralization in these tissues. A number of investigations continuing in the author's laboratory utilizing high spatial resolution x-ray microanalysis and anhydrous methods of specimen preparation are described, including aspects concerning the derivation of calibration curves from synthetic calcium phosphate solids, qualitative and quantitative analyses of calcium and phosphorus in bone from embryonic chicks and in growth plate cartilage from rats, and the role of organically-bound phosphorus in mineralizing tissues. The data obtained have helped identify brushite, CaHPO4-2H2O, as the major crystalline solid phase of calcium phosphate in the earliest mineral deposits of bone tissue, brushite and poorly crystalline hydroxyapatite in bone mineral of increasing age, and poorly crystalline hydroxyapatite in the most mature mineral portions of the tissue. Growth plate cartilage examination has revealed calcium and phosphorus in single mitochondrial granules within chondrocytes and in certain extracellular particles distinct from matrix vesicles. These results have provided important information about the possible roles of cells, extracellular components, and the organic matrix in the regulation of mineralization and about the composition, structure, and organization of the mineral phase as a function of progressively increasing age and maturation of the tissues studied.

Animals↗

PubMed1976

Ultrastructural study of lesions induced in rabbit cornea by Herpes Simplex virus types 1 and 2.

Herpes simplex virus types 1 and 2 were used to infect rabbit corneas (without abrasion), and the resulting lesions were compared by light and electron microscopy. The type 1-induced lesion developed more quickly; the infected epithelial cells underwent a progression of defined morphological changes leading to organelle destruction, cell rounding, and death. Virus particles were abundant in the nuclei and cytoplasm of all types of cell in the epithelium, in intercellular spaces, and on the basement membrane. Occasionally, virus was seen to replicate within stromal fibroblasts. In contrast, type 2, although causing similar changes in the epithelial cells, was relatively inefficient in the production of mature virions. Thus type 2 nucleocapsids were synthesized within nuclei in numbers comparable to those of types 1, but intact cytoplasmic virions were uncommon, apparently because of degradation of type 2 virions in cytoplasmic vesicles or tubules. Extracellular type 2 particles were rarely seen. This finding offers an explanation for the relatively slow development of the type 2-induced lesion, but not for its greater severity. All epithelial cells appeared to be capable of supporting replication of either type of virus.

Animals↗

PubMed2026

Tumor-associated macrophages display differential protein cargo sorting in extracellular vesicles associated with poor survival in ovarian cancer.

Ovarian cancer (OC) progression and metastasis are promoted by ascites, which constitutes a central part of the tumor microenvironment (TME). In this fluid, tumor-associated macrophages (TAMs) represent a prominent immune cell type. In addition to tumor and other host cells such as TAMs, ascites is highly enriched in soluble factors as well as extracellular vesicles (EVs). How TAMs contribute to the EV compartment of the OC TME remains, however, underexplored. In this work peripheral blood monocytes from healthy donors were differentiated into monocyte-derived macrophages (MDMs) and polarized into classically activated (M1-like), alternatively activated (M2-like) and TAM-like (by ascites incubation). For all subtypes, serum-free conditioned medium was collected for 24 h and EVs were isolated and characterized by nano-flow cytometry (nFC), label-free mass spectrometry-based proteomics and electron microscopy, among others. Our results demonstrated distinct traits for EV release and cargo across the different macrophage subtypes. Specifically, TAM-like macrophages exhibited impaired release of small EVs and reduced frequency of tetraspanin-positive particles. These EV subpopulations displayed sizing profiles closer to M1-like than to M2-like samples. Also, the low EV release in TAM-like MDMs was accompanied by altered expression of biogenesis-related markers like flotillin-1 (FLOT1) and a decreased N-glycosylation of CD63 protein, which was validated in patient-derived samples. Remarkably, the EV-associated proteome of TAMs displayed significant enrichment in both pro- and anti-inflammatory molecules with clinical value. Markers significantly enriched in the ascites TAM-EV signature were mostly associated with poor prognosis, whereas M1-like EV-related markers (pro-inflammatory) were mostly associated with longer survival. Our results confirmed previous data for proteins like CD163 and MRC1 to be associated to TAM-EVs, while also describing novel candidates with diagnostic (i.e., COLEC12) and/or prognostic (i.e., MSR1) value in plasma. Taken together, our data support a unique secretory profile of TAMs in OC and provide new EV-associated biomarkers with translational impact. Our results pave the way for a better understanding of the mechanisms behind TAM-EV cargo loading and function, and how these cells participate in the TME landscape.

Humans↗

PubMed2025

Examining the bacterial diversity including extracellular vesicles in air and soil: implications for human health.

As the significance of human health continues to rise, the microbiome has shifted its focus from microbial composition to the functional roles it plays. In parallel, interest in ultrafine particles associated with clinically important impact has been increasing. Bacterial extracellular vesicles (BEVs), involved in systemic microbiome activity, are nano-sized spherical vesicles (20 - 100 nm in diameter) containing DNA, RNA, proteins, and lipids. They are known to be absorbed into the body potentially through air and soil, circulate in the blood, and directly impact diseases by affecting organs. Therefore, the aim of this study is to examine the biodiversity of bacteria and BEVs and predicted functional pathways. We sampled air and soil samples in Seoul, Korea and analyzed metagenomics based on 16S rRNA sequencing. At the phylum levels, Firmicutes in BEVs from soil and air were significantly higher than in bacteria, and Acidobacteria in both bacteria and BEVs from soil were significantly higher than from air (p < 0.05). The most dominant genera were Pseudomonas in bacteria from air and soil; and Escherichia-Shigella in BEVs from air and soil. In addition, Two-component system (ko02020) and ATP-binding cassette transporters (ko02010) were dominant functional pathways in both air and soil. The most functional pathways and orthologous groups were significantly different between air and soil (p < 0.05). In conclusion, human health can be affected differently depending on type of environment. Future study is necessary to have a better understanding of human health effects from environmental microbiota.

Soil Microbiology↗

PubMed1979

Calcification of aging articular cartilage in man.

Calcification of the articular cartilage was studied ultrastructually using normal femoral heads obtained from necropsies of persons ranging in age from 11 months to 80 years. Mineral crystals which appeared during the initial stages of deposition were morphologically divided into two types. Type A crystals were slender, twisted and curved, measuring from 100 nm to 360 nm in length. Type B crystals were short, needle-like and slightly curved, measuring from 30 nm to 160 nm in length. Type A crystals were found mainly in the developing epiphysis during childhood. Type B crystals were generally found in the calcified zone of adult articular cartilage. Both types of crystals initially appeared in close proximity to extracellular membrane-invested electron dense particle called "matrix vesicles", and gradually increased in number to form calcified cartilage matrix. The morphological differences between type A and B crystals might be caused by biochemical alterations of the cartilage matrices and/or biomechanical changes in the joints of children and adults.

Adolescent↗

PubMed1979

Partition of alkane by an extracellular vesicle derived from hexadecane-grown Acinetobacter.

The enhanced solubility of hexadecane in the growth medium of hexadecane-grown Acinetobacter species has been related to the accumulation of an extracellular vesicular component. The partition of hexadecane was determined by measuring the amount of [3H]hexadecane bound to the vesicular particle. The vesicle was characterized as a phospholipid-rich, lipopolysaccharide-rich particle with a polypeptide composition similar to the outer membrane of Acinetobacter. The accumulation of an extracellular vesicular component that binds hexadecane in the form of a microemulsion represents another example of molecules produced by microorganisms in response to paraffinic substrates.

Acinetobacter↗

PubMed1978

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.

Bacterial Chromatophores↗

PubMed1979

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.).

Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freeze-fracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.

Animals↗

PubMed1979

Yolk transport in the ovarian follicle of the hen (Gallus domesticus): lipoprotein-like particles at the periphery of the oocyte in the rapid growth phase.

Thin sections of the oocyte periphery and surrounding granulosa layer from 1--5 day preovulatory follicles were examined by transmission electron microscopy. With the use of certain procedures in tissue preparation, notably the tannic acid method, numerous particles in the range of 15--40 nm with a mean diameter of 27 nm were observed in both extra- and intracellularly. The particles were abundant in the granulosa basal lamina, in the spaces between the granulosa cells and in the perivitelline space. They appeared to adhere to the oolemma as a continuous double layer which was also observed to line the coated vesicles, 200--350 nm in diameter, invaginating from the oolemma. The layer of particles was not found on the plasma membranes of the granulosa cells, nor were particles present within the cells. In the peripheral cytoplasm of the oocyte the yolk spheres, ranging upwards from 250 nm diameter, were membrane-bound and contained tightly packed particles similar to those on the oolemma. Bodies displaying features intermediate between coated vesicles and yolk spheres suggested that, on entry into the cell, loss of the cytoplasmic coat and obliteration of the vesicular lumen gave rise to nascent yolk spheres which then fused together to form the larger spheres. The extracellular layer, coated vesicles and smaller yolk spheres were absent in oocytes fixed after a 10-min delay. The evidence indicated that 27-nm particles were transferred from the basal lamina to the oocyte surface via the intergranulosa cell channels, incorporated into the cell by adsorptive endocytosis and then transferred to the yolk spheres with little morphological alteration. The identity of the particles with very low density lipoproteins, the major components of the yolk solids, was discussed.

Animals↗

PubMed1979

Pseudoviral hollow-cored vesicles in multiple sclerosis brain.

Novel, superficially 'virus-like' hollow-cored particles 50--60 nm in diameter were found in the perivascular extracellular space of the brain from a patient who died with acute multiple sclerosis (MS). It is concluded that they are not virions but are derived from myelin undergoing vesicular demyelination. This case demonstrates the need for caution in the interpretation of unusual electron microscopic appearances.

Acute Disease↗

PubMed1978

C-type virus-lymphocyte interactions in developing mouse thymus.

The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.

Animals↗

PubMed1979

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone.

Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by the freeze-fracture technique. When BWSV was applied for short periods (10-15 min) in the presence of Ca2+, numerous dimples (P face) or protuberances (E face) appeared on the presynaptive membrane and approximately 86% were located immediately adjacent to the double rows of large intramembrane particles that line the active zones. When BWSV was applied for 1 h in the presence of Ca2+, the nerve terminals were depleted of vesicles, few dimples or protuberances were seen, and the active zones were almost completely disorganized. The P face of the presynaptic membrane still contained large intramembrane particles. When muscles were soaked for 2-3 h in Ca2+-free solutions, the active zones became disorganized, and isolated remnants of the double rows of particles were found scattered over the P face of the presynaptic membrane. When BWSV was applied to these preparations, dimples or protuberances occurred almost exclusively alongside disorganized active zones or alongside dispersed fragments of the active zones. The loss of synaptic vesicles from terminals treated with BWSV probably occurs because BWSV interferes with the endocytosis of vesicle membrane. Therefore, we assume that the dimples or protuberances seen on these terminals identify the sites of exocytosis, and we conclude that exocytosis can occur mostly in the immediate vicinity of the large intramembrane particles. Extracellular Ca2+ seems to be required to maintain the grouping of the large particles into double rows at the active zones, but is not required for these particles to specify the sites of exocytosis.

Animals↗

PubMed1978

Changes in the synapses of spiral ganglion cells in the rostral anteroventral cochlear nucleus of the waltzing guinea pig following hair cell loss.

Between 10 and 60 days of age in the waltzing guinea pig, there is a genetically induced loss of all hair cells in the organ of Corti. About 43% of the spiral ganglion cells degenerate between 30 and 60 days of age. After 90 days of age, there is no further loss of spiral ganglion cells. Both Type I and II ganglion cells remain and are without afferent input. The terminals of these ganglion cells in the rostral AVCN, the end bulbs of Held, are normal until 30 days of age. During the period of ganglion cell loss degenerating end bulbs are seen. After 60 days of age, when most ganglion cell degeneration is complete, the remaining end bulbs have fewer synaptic vesicles and their synaptic junctions are flattened. The channels of enlarged extracellular space, which normally surround each synaptic junction or small groups of junctions, are only infrequently present. In freeze-fracture replicas of the rostral AVCN of waltzing guinea pigs after hair cell loss, the number of large, non-aggregate particles on the external leaflet of the principal cell opposite the end bulb is increased, and the number of perisynaptic aggregates is decreased compared to waltzing guinea pigs 10 days of age. The junctional aggregates are unaltered. These changes in the presynaptic terminal and postsynaptic membrane may be related to the loss of afferent input to the spiral ganglion cells, suggesting that activity is important for maintaining the synapse.

Age Factors↗

PubMed2026

The present and future of nonviral delivery-based genome editing for hereditary hearing loss.

PURPOSE OF REVIEW: This review summarizes nonviral genome-editing delivery platforms for hereditary hearing loss, focusing on lipid nanoparticles (LNPs) and engineered virus-like particles (eVLPs), and discusses their advantages over adeno-associated virus-based delivery, as well as the barriers to clinical translation. RECENT FINDINGS: Recent advances have established LNPs as a clinically advanced nonviral platform, although challenges related to inner ear biodistribution, cell type specificity, endosomal escape, and immunogenicity remain to be addressed. In parallel, eVLPs have undergone substantial technical evolution, progressing from early low efficiency systems to advanced base editor- and prime editor-eVLP architectures that enhance cargo loading and editing efficiency. Extracellular vesicle-based genome editing has also emerged as an additional platform, although issues related to reproducibility, loading efficiency, and scalability remain major hurdles. SUMMARY: Nonviral genome editing platforms expand the therapeutic toolkit for hereditary hearing loss by enabling transient delivery of genome editors with potential safety advantages. Future efforts should focus on characterizing biodistribution and immunogenicity, refining cell type-specific tropism, and establishing scalable manufacturing processes to enable successful clinical translation.

Humans↗

PubMed1978

Cell membrane structure of human giant-celled glioblastoma.

A giant-cell glioblastoma was examined by electron microscopy and by the freeze-fracture technique. The cell membranes bordering the extensive extracellular space often showed complicated undulations and peripheral vacuoles as well as occasional microvilli or filopodia. The undulations were mainly composed of plasmalemmal vesicles as well as of large (400--800 nm in diameter) and small (30--50 nm in diameter) localized protrusions and invaginations of the cell membrane. Deep invaginations of the cell membrane apparently resulted in two separate cytoplasmic portions. Locking of protruded cytoplasmic tongues and adherens junctions were sometimes seen in closely approximated cell membranes. The average number of membrane particles per micrometer2 was 630 +/- 130 on the P face and 180 +/- 30 on the E face. The membrane particles were occasionally aggregated to form clusters about 30 to 150 micrometer in diameter. Gap junctions were occasionally found, but there were no tight junctions. Large particles about 30 nm in diameter were found in places.

Cell Membrane↗