Analyzing retraction responses of Tubifex tubifex (Oligochaeta: Naididae) colonies to copper treatments through a digital image analysis approach for developing early biological warning systems.

Biological monitoring and assessments are commonly used for sustainable ecosystem management. Oligochaetes are found in various freshwater ecosystems and have been used as indicators of water quality and for the biological assessment of aquatic ecosystems. Among aquatic oligochaetes, the sludge worm Tubifex tubifex (Oligochaeta, Naididae) is tolerant to organic pollution and has been used as a biomonitoring indicator of toxicity and organic pollution. In this study, we investigated the response of worm colonies to copper (CuSO4) treatments (0.01, 0.05, 0.1, 0.5, and 1.0 mg/L) in an observation cage (100 mL beaker) for 30 min. Using a digital image analysis approach, we measured the changes in the colony image area between pre- and post-copper treatment. After copper treatment, the colony image area tended to decrease, even at low copper concentrations. In addition, the colony areas did not recover to their original levels at high concentrations, although those at low concentrations did. Area decreased proportional to the logarithm of the copper concentration. Finally, our results present the possible use of the retraction responses of Tubifex tubifex colonies to chemical disturbances as early biological warning systems.

Tunable structure of chimeric isomaltomegalosaccharides with double α-(1 → 4)-glucosyl chains enhances the solubility of water-insoluble bioactive compounds.

Isomaltomegalosaccharides with α-(1 â†’ 4) and α-(1 â†’ 6)-segments solubilize water-insoluble ligands since the former complexes with the ligand and the latter solubilizes the complex. Previously, we enzymatically synthesized isomaltomegalosaccharide with a single α-(1 â†’ 4)-segment at the reducing end (S-IMS) by dextran dextrinase (DDase), but the chain length [average degree of polymerization (DP) ≤ 9] was insufficient for strong encapsulation. We hypothesized that the conjugation of longer α-(1 â†’ 4)-segment afforded the promising function although DDase is incapable to do so. In this study, the cyclodextrin glucanotransferase-catalyzed coupling reaction of α-cyclodextrin to S-IMS synthesized a new α-(1 â†’ 4)-segment at the nonreducing end (N-4S) of S-IMS to form D-IMS [IMS harboring double α-(1 â†’ 4)-segments]. The length of N-4S was modulated by the ratio between α-cyclodextrin and S-IMS, generating N-4Ss with DPs of 7-50. Based on phase-solubility analysis, D-IMS-28.3/13/3 bearing amylose-like helical N-4S with DP of 28.3 displayed a water-soluble complex with aromatic drugs and curcumin. Small-angle X-ray scattering revealed the chain adapted to rigid in solution in which the radius of gyration was estimated to 2.4 nm. Furthermore, D-IMS with short N-4S solubilized flavonoids of less-soluble multifunctional substances. In our research, enzyme-generated functional biomaterials from DDase were developed to maximize the hydrophobic binding efficacy towards water-insoluble bioactive compounds.

Complete mitochondrial genome of the decomposer Ptecticus tenebrifer Walker, 1849 (Insecta: Diptera: Stratiomyidae).

Ptecticus tenebrifer (Walker, 1849), which is endemic in East Asia, plays a critical role in natural processing of organic waste. In this study, we found that the complete mitochondrial genome of P. tenebrifer is 15681 bp in length obtained from the MGIseq system (MGI) with 150 bp paired-end reads. As with typical animal mitochondrial genomes, it contains 13 protein coding genes, 22 tRNA genes, and 2 rRNA genes. The phylogenetic analysis was performed using the maximum likelihood method with a bootstrap value of 500 and the Tamura-Nei model and reveals the close relationship with P. aurifer.

Partial depolymerization of tamarind seed xyloglucan and its functionality toward enhancing the solubility of curcumin.

Polysaccharides of tamarind seed, a byproduct of the tamarind pulp industry, displayed a potential solubility improvement of lipophilic bioactive molecules but their textural characteristics hinder the dietary formulation. In contrast, the commonly available xyloglucan oligosaccharides (XOSs) with degrees of polymerization (DPs) of 7, 8, and 9 were too short to maintain their ability. The binding capacity of the between sizes is unknown due to a lack of appropriate preparation. We prepared xyloglucan megalosaccharides (XMSs) by partial depolymerization, where term megalosaccharide (MS) defines the middle chain-length saccharide between DPs 10 and 100. Digestion with fungal cellulase enabled reproducible active XMSs. Further identification of pure XMS segments indicated that XMS-B has an average DP of 17.2 (Gal3Glc8Xyl6) with a branched dimer of XOS 8 and 9 and was free of side-chain arabinose, the residue influencing high viscosity. Curcumin, a bioactive pigment, has poor bioavailability because of its water insolubility. XMSs with average DPs of 15.4-24.3 have similarly sufficient capacities to solubilize curcumin. The solubility of curcumin was improved 180-fold by the addition of 50 %, w/v, XMSs, which yielded a clear yellow liquid. Our findings indicated that XMSs were a promising added-value agent in foods and pharmaceuticals for the oral intake of curcumin.

Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes.

Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 â†’ 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine.

Red cell distribution width as a prognosticator in patients with heart failure.

AIMS: Increased red cell distribution width (RDW) is a poor prognostic factor in patients with heart failure (HF). However, only a few large-scale studies have identified the clinical utility of RDW after adjusting for covariates affecting RDW. METHODS AND RESULTS: From January 2010 to April 2021, we retrospectively enrolled patients diagnosed with HF from three referral hospitals with available RDW data (taken within 3 months of HF diagnosis) using an integrated clinical data system. Patients with an ejection fraction (EF) < 50% or HFA-PEFF (Heart Failure Association Pre-test assessment, Echocardiography and natriuretic peptide, Functional testing, Final aetiology) score ≥ 2 without severe valvular heart disease or coronary revascularization were enrolled. The primary endpoint was all-cause mortality, and cardiovascular mortality was also collected. Multivariable Cox regression analysis and stabilized inverse probability of treatment weighting (IPTW) were used to identify any association between RDW and all-cause death by balancing covariates or compounding factors. The global χ2 score was calculated and discrimination analysis was performed to evaluate the incremental value of RDW in predicting prognosis. Among the 6599 participants enrolled in this study, 1256 (19.0%) cases of all-cause death occurred, and the median duration of follow-up was 887 (interquartile range 351-1589) days. Elevated RDW at the initial diagnosis was associated with poor prognosis [cumulative incidence: 819 (30.2%) vs. 437 (11.2%), relative risk 1.58, 95% confidence interval (CI) 1.51-1.67, log-rank P < 0.001]. Multivariable Cox analysis showed that elevated RDW was a poor prognostic factor for the primary endpoint [hazard ratio (HR) 1.11, 95% CI 1.06-1.16, P < 0.001], independent of clinical risk factors, N-terminal pro-brain natriuretic peptide (NT-proBNP), and EF, which was concordant with the stabilized IPTW (HR 1.29, 95% CI 1.10-1.49, P < 0.001). Adding RDW to model composed of traditional risk factors, NT-proBNP, and echocardiographic parameters showed incremental prognostic value for predicting poor prognosis (area under the receiver operating characteristic curve, 0.799-0.826; P < 0.001). CONCLUSIONS: Increased RDW at the time of diagnosis is associated with poor prognosis in patients with HF, independent of clinical risk factors, such as NT-proBNP, and echocardiographic parameters. Therefore, RDW may aid in the management of these patients beyond traditional risk factors.

Discovery of α-l-Glucosidase Raises the Possibility of α-l-Glucosides in Nature.

Glucose, a common monosaccharide in nature, is dominated by the d-enantiomer. Meanwhile, the discovery of l-glucose-utilizing bacteria and the elucidation of their metabolic pathways 10 years ago suggests that l-glucose exists naturally. Most carbohydrates exist as glycosides rather than monosaccharides; therefore, we expected that nature also contains l-glucosides. Sequence analysis within glycoside hydrolase family 29 led us to identify two α-l-glucosidases, ClAgl29A and ClAgl29B, derived from Cecembia lonarensis LW9. ClAgl29A and ClAgl29B exhibited higher K m, k cat, and k cat/K m values for p-nitrophenyl α-l-glucoside than that for p-nitrophenyl α-l-fucoside. Structural analysis of ClAgl29B in complex with l-glucose showed that these enzymes have an active-site pocket that preferentially binds α-l-glucoside, but excludes α-l-fucoside. These results suggest that ClAgl29A and ClAgl29B evolved to hydrolyze α-l-glucoside, implying the existence of α-l-glucoside in nature. Furthermore, α-l-glucosidic linkages (α-l-Glc-(1 → 3)-l-Glc, α-l-Glc-(1 → 2)-l-Glc, and α-l-Glc-(1 → 6)-l-Glc) were synthesized by the transglucosylation activity of ClAgl29A and ClAgl29B. We believe that this study will lead to new research on α-l-glucosides, including determining the physiological effects on humans, and the discovery of novel α-l-glucoside-related enzymes.

Animal Welfare Assessment in 16 Zoos in South Korea Using the Modified Animal Welfare Assessment Grid.

Various assessment tools that have been proposed thus far have disadvantages in that they are complex, time-consuming, non-objective, and not convenient for assessing multiple zoos. This study aimed to develop a simple, objective, and reliable welfare assessment tool, the modified Animal Welfare Assessment Grid (AWAG), that can be applied in South Korea, where there is no licensing system for zoos. The AWAG has four main sections: physical, psychological, environmental, and procedural. These four sections include 23 welfare factors like general conditions, behaviors, housing, and restraints, for which each individual or group of animals is given a score. The modified AWAG system was applied by converting the 10-point rating scale of the original AWAG to a 6-point Likert scale. Sixteen zoos in Korea were selected based on the zoos with the most animals. Three inspectors assessed the scores of each animal and then averaged the results. The total data surveyed included 16,065 items. Zoos were largely classified into four grades based on the size of the zoo, animal species, and operating organization. In a relatively short period of 14 days, all the zoos were successfully assessed. Despite the shortened and modified assessment tool, the inter-rater reliability among inspectors was 0.942 with high objectivity. The modified AWAG could identify welfare differences between grades of Korean zoos. There were large differences between zoos in most environmental sections and some zoos were evaluated as having inadequate welfare levels. The modified AWAG showed high usability and objectivity. In addition, it was possible to determine which environmental or procedural sections could potentially help improve physical and psychological scores. The modified AWAG is an objective method that could set the direction for the improvement of zoo welfare in the future.

Characteristic functional cores revealed by hyperbolic disc embedding and k-core percolation on resting-state fMRI.

Hyperbolic disc embedding and k-core percolation reveal the hierarchical structure of functional connectivity on resting-state fMRI (rsfMRI). Using 180 normal adults' rsfMRI data from the human connectome project database, we visualized inter-voxel relations by embedding voxels on the hyperbolic space using the [Formula: see text] model. We also conducted k-core percolation on 30 participants to investigate core voxels for each individual. It recursively peels the layer off, and this procedure leaves voxels embedded in the center of the hyperbolic disc. We used independent components to classify core voxels, and it revealed stereotypes of individuals such as visual network dominant, default mode network dominant, and distributed patterns. Characteristic core structures of resting-state brain connectivity of normal subjects disclosed the distributed or asymmetric contribution of voxels to the kmax-core, which suggests the hierarchical dominance of certain IC subnetworks characteristic of subgroups of individuals at rest.

Carbohydrate-binding module of cycloisomaltooligosaccharide glucanotransferase from Thermoanaerobacter thermocopriae improves its cyclodextran production.

Thermoanaerobacter thermocopriae-derived thermostable cycloisomaltooligosaccharide (CI)-forming enzymes catalyze the production of CIs from dextran. The primary structure of the enzyme is comprised of CI glucanotransferase (TtCITase) at the N-terminal region and long isomaltooligosaccharide-forming enzyme (TtTGase) at the C-terminal region connected by carbohydrate-binding module family 35 (CBM, TtCBM). Three truncated mutants of CI-forming enzymes were successfully produced in Corynebacterium glutamicum, a food-grade host system, and their biochemical properties were characterized. The enzymes had optimum at pH 6.0 and pH-stability (5.0-12.0). Three enzymes had optimum temperature over 55 °C and they maintained 80% activity at 55 °C for 2 h, 12 h, and 18 h, respectively. Enzymes without CBM showed weaker allosteric behavior than those of other enzymes, which suggests the important role of CBM in allosteric behavior. However, CBM bearing enzymes showed high production of CIs with various degree of polymerization. These enzymes have potential application as the encapsulating material for insoluble pharmaceutical biomaterials.

Alterations in Social Brain Network Topology at Rest in Children With Autism Spectrum Disorder.

OBJECTIVE: Underconnectivity in the resting brain is not consistent in autism spectrum disorder (ASD). However, it is known that the functional connectivity of the default mode network is mainly decreased in childhood ASD. This study investigated the brain network topology as the changes in the connection strength and network efficiency in childhood ASD, including the early developmental stages. METHODS: In this study, 31 ASD children aged 2-11 years were compared with 31 age and sex-matched children showing typical development. We explored the functional connectivity based on graph filtration by assessing the single linkage distance and global and nodal efficiencies using resting-state functional magnetic resonance imaging. The relationship between functional connectivity and clinical scores was also analyzed. RESULTS: Underconnectivities within the posterior default mode network subregions and between the inferior parietal lobule and inferior frontal/superior temporal regions were observed in the ASD group. These areas significantly correlated with the clinical phenotypes. The global, local, and nodal network efficiencies were lower in children with ASD than in those with typical development. In the preschool-age children (2-6 years) with ASD, the anterior-posterior connectivity of the default mode network and cerebellar connectivity were reduced. CONCLUSION: The observed topological reorganization, underconnectivity, and disrupted efficiency in the default mode network subregions and social function-related regions could be significant biomarkers of childhood ASD.

Hyperbolic disc embedding of functional human brain connectomes using resting-state fMRI.

The brain presents a real complex network of modular, small-world, and hierarchical nature, which are features of non-Euclidean geometry. Using resting-state functional magnetic resonance imaging, we constructed a scale-free binary graph for each subject, using internodal time series correlation of regions of interest as a proximity measure. The resulting network could be embedded onto manifolds of various curvatures and dimensions. While maintaining the fidelity of embedding (low distortion, high mean average precision), functional brain networks were found to be best represented in the hyperbolic disc. Using the 𝕊1/ℍ2 model, we reduced the dimension of the network into two-dimensional hyperbolic space and were able to efficiently visualize the internodal connections of the brain, preserving proximity as distances and angles on the hyperbolic discs. Each individual disc revealed relevance with its anatomic counterpart and absence of center-spaced node. Using the hyperbolic distance on the 𝕊1/ℍ2 model, we could detect the anomaly of network in autism spectrum disorder subjects. This procedure of embedding grants us a reliable new framework for studying functional brain networks and the possibility of detecting anomalies of the network in the hyperbolic disc on an individual scale.

Enhanced biotransformation of the minor ginsenosides in red ginseng extract by Penicillium decumbens ß-glucosidase.

Compound K (C-K) and Rh2, which are present at low levels in ginseng and ginseng extracts, have higher intestinal absorption rates than other ginsenosides. Here, we attempted to convert ginsenoside Rb1 to C-K using a ß-glucosidase from Penicillium decumbens. Ten commercially available enzymes were screened to identify enzymes that can convert ginsenoside Rb1 to C-K, resulting in the selection of a P. decumbens-derived ß-glucosidase. ß-Glucosidase showed maximum activity at pH 4.0 and 60 °C; its substrate specificity for ginsenoside Rb1 was investigated. The main glucoside-hydrolyzing pathways were as follows: ginsenoside Rb1 or Rd → gypenoside XVII → F2 → C-K and ginsenoside Rg3 → Rh2. The P. decumbens-derived ß-glucosidase was used to generate C-K and Rh2 using protopanaxadiol-type ginsenosides as substrates. Additionally, to apply this enzyme to the commercialized red ginseng extract products, the contents of C-K and Rh2 in the total ginsenosides significantly (p < 0.05) increased up to 36-fold and 8.9-fold, respectively, higher than prior to subjecting to biotransformation. To the best of our knowledge, this is the first report of the dual biotransformation of C-K and Rh2 by a food-grade commercial enzyme. This study demonstrates that the use of a specific ß-glucosidase may increase C-K and Rh2 contents in the ginseng extract through a simple biotransformation process and, thus, enhance its health benefits.

Synthesis and characteristics of a rebaudioside-A like compound as a potential non-caloric natural sweetener by Leuconostoc kimchii dextransucrase.

Stevioside (ST) is currently considered as a highly-demanded natural and zero-caloric replacer of sucrose with several health-promoting properties. Nonetheless, its bitter aftertaste limits its use in the food industry. Herein, glucosyl steviosides were synthesized using primarily a food-grade lactic acid bacteria, Leuconostoc kimchii dextransucrase and conversion yield (%) was 40.3%. A glucose moiety was transferred stereo-selectively to ST by α-1,6-linkage and this is the first report about obtaining rebaudioside A (Reb-A) like glucosyl stevioside-2 (STG-2). Glucosyl steviosides revealed greatly improved stability up to 120 °C and remained stable over 32.1% and 58.12% in the pH (1.4) compared with 30.55% of ST. Moreover, the glucosylated steviosides improved the stability, reaching 95% after 30 days and Reb-A like compound (STG-2) especially exhibited higher stability in commercial beverages. Furthermore, the glucosyl steviosides showed over 1.92- and 2.24-fold decreases than that of enzymatically modified ST in the glucose generation rate test.

Extracellular Vesicles Generated Using Bioreactors and their Therapeutic Effect on the Acute Kidney Injury Model.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.

Nutritional Management in a Patient with Citrullinemia Type 1.

For patients with citrullinemia type 1, nutritional management is essential to prevent the occurrence of complications associated with hyperammonemia. This report describes a patient who had been receiving nutrition intervention for more than 3 years. A newborn diagnosed with hyperammonemia due to citrullinemia visited Ajou University Hospital and was referred to the nutrition team. After receiving acute treatment, the infant was regularly fed with specialized formula. A protein-restricted diet is recommended for maintaining normal development and achieving long-term survival. Through continuous provision of nutritional intervention, the child showed normal growth and development, and the energy-protein supply was maintained appropriately. This case clearly shows the importance of medical nutrition therapy for patients with citrullinemia.

Cutaneous T-cell lymphoma in Asian patients: a multinational, multicenter, prospective registry study in Asia.

Cutaneous T-cell lymphomas (CTCLs) are a group of T-cell lymphomas with low incidence. Due to their indolent characteristics, treatment strategies have not yet been established for advanced CTCLs. In this study, relative incidence of CTCLs in Asia was estimated and the therapeutic outcomes presented based on various treatments currently used in clinics for advanced CTCLs. As part of a prospective registry study of peripheral T-cell lymphoma (PTCL) conducted across Asia, including Korea, China, Taiwan, Singapore, Malaysia, and Indonesia, subgroup analysis was performed for patients with CTCLs. Among 486 patients with PTCL, 37 with CTCL (7.6%) were identified between April 2016 and February 2019. Primary cutaneous ALK-negative anaplastic large cell lymphoma (ALCL, 35.1%) was the most common subtype. With a median follow-up period of 32.1 months, median progression-free survival (PFS) was 53.5 months (95% CI 0.0-122.5), and overall survival was not reached. 14 patients (48.2%) underwent subsequent treatment after the first relapse, but the response rate was 20% with a PFS of 2.2 months (95% CI 0.3-4.0). Six patients received autologous stem cell transplantation (auto-SCT). However, auto-SCT did not result in better outcomes. Additional studies are needed on standard care treatment of advanced or refractory and relapsed CTCLs.

Multifluorescence Single Extracellular Vesicle Analysis by Time-Sequential Illumination and Tracking.

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

The adipokine Retnla deficiency increases responsiveness to cardiac repair through adiponectin-rich bone marrow cells.

Resistin-like alpha (Retnla) is a member of the resistin family and known to modulate fibrosis and inflammation. Here, we investigated the role of Retnla in the cardiac injury model. Myocardial infarction (MI) was induced in wild type (WT), Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiac function was assessed by echocardiography and was significantly preserved in the KO mice, while worsened in the TG group. Angiogenesis was substantially increased in the KO mice, and cardiomyocyte apoptosis was markedly suppressed in the KO mice. By Retnla treatment, the expression of p21 and the ratio of Bax to Bcl2 were increased in cardiomyocytes, while decreased in cardiac fibroblasts. Interestingly, the numbers of cardiac macrophages and unsorted bone marrow cells (UBCs) were higher in the KO mice than in the WT mice. Besides, phosphorylated histone H3(+) cells were more frequent in bone marrow of KO mice. Moreover, adiponectin in UBCs was notably higher in the KO mice compared with WT mice. In an adoptive transfer study, UBCs were isolated from KO mice to transplant to the WT infarcted heart. Cardiac function was better in the KO-UBCs transplanted group in the WT-UBCs transplanted group. Taken together, proliferative and adiponectin-rich bone marrow niche was associated with substantial cardiac recovery by suppression of cardiac apoptosis and proliferation of cardiac fibroblast.

Single-vesicle imaging and co-localization analysis for tetraspanin profiling of individual extracellular vesicles.

Extracellular vesicles (EVs) are secreted nano-sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal-reflection microscopy and analyzes their co-localization to investigate EV subpopulations. The single-vesicle imaging and co-localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.