Increased expression of six-large extracellular vesicle-derived miRNAs signature for nonvalvular atrial fibrillation.

BACKGROUNDS: Non-valvular atrial fibrillation (AF) is the most common type of cardiac arrhythmia. AF is caused by electrophysiological abnormalities and alteration of atrial tissues, which leads to the generation of abnormal electrical impulses. Extracellular vesicles (EVs) are membrane-bound vesicles released by all cell types. Large EVs (lEVs) are secreted by the outward budding of the plasma membrane during cell activation or cell stress. lEVs are thought to act as vehicles for miRNAs to modulate cardiovascular function, and to be involved in the pathophysiology of cardiovascular diseases (CVDs), including AF. This study identified lEV-miRNAs that were differentially expressed between AF patients and non-AF controls. METHODS: lEVs were isolated by differential centrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), flow cytometry and Western blot analysis. For the discovery phase, 12 AF patients and 12 non-AF controls were enrolled to determine lEV-miRNA profile using quantitative reverse transcription polymerase chain reaction array. The candidate miRNAs were confirmed their expression in a validation cohort using droplet digital PCR (30 AF, 30 controls). Bioinformatics analysis was used to predict their target genes and functional pathways. RESULTS: TEM, NTA and flow cytometry demonstrated that lEVs presented as cup shape vesicles with a size ranging from 100 to 1000 nm. AF patients had significantly higher levels of lEVs at the size of 101-200 nm than non-AF controls. Western blot analysis was used to confirm EV markers and showed the high level of cardiomyocyte expression (Caveolin-3) in lEVs from AF patients. Nineteen miRNAs were significantly higher (> twofold, p < 0.05) in AF patients compared to non-AF controls. Six highly expressed miRNAs (miR-106b-3p, miR-590-5p, miR-339-3p, miR-378-3p, miR-328-3p, and miR-532-3p) were selected to confirm their expression. Logistic regression analysis showed that increases in the levels of these 6 highly expressed miRNAs associated with AF. The possible functional roles of these lEV-miRNAs may involve in arrhythmogenesis, cell apoptosis, cell proliferation, oxygen hemostasis, and structural remodeling in AF. CONCLUSION: Increased expression of six lEV-miRNAs reflects the pathophysiology of AF that may provide fundamental knowledge to develop the novel biomarkers for diagnosis or monitoring the patients with the high risk of AF.

Molecular properties and ligand specificity of zebrafish intelectin-2.

Intelectins are immune lectins expressed in chordates, including several fish species, in which intelectins are known to be upregulated upon infection. However, the basic biochemical properties and bacteria binding specificities of several fish intelectins are not well studied. We focus our investigation on zebrafish intelectin-2 (DrIntL-2) that is predominantly expressed in the gastrointestinal tract. The disulfide-linked oligomeric state and the cysteine responsible for intermolecular disulfide bonds are identified. DrIntL-2 is a globular particle of around 30 nm. In addition to the typical exocyclic 1,2-diol ligands, DrIntL-2 binds ß-1,3-glucan and recognizes Salmonella typhimurium and Pseudomonas aeruginosa. This investigation not only shed light on the fish innate immunity that will be essential for the aquaculture industry, but will also provide a foundation for further application of DrIntL-2 in bacteria detection and identification.

Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2.

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including "cell-cell adhesion," "cell-cell adherens junction," "mitochondrial inner membrane," "endoplasmic reticulum chaperone complex," and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

ECM-Body: A Cell-Free 3D Biomimetic Scaffold Derived from Intact Planarian Body.

Extracellular matrix (ECM) plays key roles in shaping fates of stem cells, not only by providing a suitable niche but also by mediating physical and biochemical cues. Despite intensive investigations on regeneration, the roles of ECM in fate determination of stem cells in animals with great regenerative potency, such as planarian, have remained unclear. Here, we developed a method for decellularizing and isolating extracellular matrix from planarians. Although the isolated scaffold appears translucent, it contains all the internal features resembling those of the structure of intact planarians, and we thus called it the "ECM-body". Nuclear staining demonstrated that the ECM-body contains very few or no remaining cells. Histological sections displayed well-preserved morphological integrity of the specimen. Scanning electron microscopy showed a porous surface on the ECM-body, potentially suitable for housing cells. Furthermore, our preliminary experiment suggested that ECM-body can be utilized as a biomimetic scaffold for cell culture as it may support survival of injected neoblasts.

Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus.

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection.

UNLABELLED: Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1-anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever. IMPORTANCE: Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs), tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patients' blood are changed in response to DENV infection. Infection of cells led to programmed cell death and MP release. In patients' blood, the majority of MPs originated from red blood cells and platelets. Decreased platelet-derived MPs were associated with a bleeding tendency, while increased levels of red blood cell-derived MPs (RMPs) correlated with more severe disease. Importantly, the level of RMPs during the early acute phase could serve as a biomarker to identify patients with potentially severe disease who require immediate care.

Lysinibacillus sphaericus binary toxin induces apoptosis in susceptible Culex quinquefasciatus larvae.

During sporulation, a Gram-positive bacterium Lysinibacillus sphaericus (Ls) produces the mosquito larvicidal binary toxin composed of 2 subunits, BinA and BinB. Full toxicity against Culex and Anopheles mosquito larvae is achieved when both subunits are administered together at equimolar amounts. Although cellular responses to Bin toxin have been reported in previous studies, it remains essential to extensively examine the cytopathic effects in vivo to define the underlying mechanism of larval death. In this study, 4th instar Culex quinquefasciatus larvae fed with different doses of Bin toxin were analyzed both for ultrastructural as well as biochemical effects. Typical morphological changes consistent with apoptosis were observed in mosquito larvae exposed to Bin toxin, including mitochondrial swelling, chromatin condensation, cytoplasmic vacuolization and apoptotic cell formation. Bin toxin also induced the activation of caspase-9 and caspase-3 in larval midgut cells. Our current observations thus suggest that Bin toxin triggers apoptosis via an intrinsic or mitochondrial pathway in vivo, possibly contributing to larval death.

Increased small extracellular vesicle levels and decreased miR-126 levels associated with atrial fibrillation and coexisting diabetes mellitus.

BACKGROUND: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia. Diabetes mellitus (DM) is one of the risk factors for the development of stroke and thromboembolism in patients with AF. Early identification may reduce the incidence of complications and mortality in AF patients. HYPOTHESIS: AF patients with DM have different pattern of small extracellular vesicle (sEV) levels and sEV-derived microRNA (miRNA) expression compared with those without DM. METHODS: We compared sEV levels and sEV-miRNA expression in plasma from AF patients with and without DM using nanoparticle tracking analysis and droplet digital polymerase chain reaction, respectively. RESULTS: We observed a significant increase in total sEV levels (p = .004) and a significant decrease in sEV-miR-126 level (p = .004) in AF patients with DM. Multivariate logistic regression analysis revealed a positive association between total sEV levels and AF with DM (p = .019), and a negative association between sEV-miR-126 level and AF with DM (p = .031). The combination of clinical data, total sEVs, and sEV-miR-126 level had an area under the curve of 0.968 (p < .0001) for discriminating AF with DM, which was shown to be significantly better than clinical data analysis alone (p = .0368). CONCLUSIONS: These results suggest that an increased level of total sEV and a decreased sEV-miR-126 level may play a potential role in the pathophysiology and complications of AF with DM, especially endothelial dysfunction, and can be considered as an applied biomarker for distinguishing between AF with and without DM.

Biochemical and ligand binding properties of recombinant Xenopus laevis cortical granule lectin-1.

Intelectins are putative innate immune lectins that are found throughout chordates. The first intelectin reported was Xenopus laevis cortical granule lectin-1 (XCGL-1 or XL-35). XCGL-1 is critical in fertilization membrane development in Xenopus. Here, we explored the biochemical properties of XCGL-1. The cysteines responsible for forming intermolecular disulfide bonds were identified. XCGL-1 adopted a four-lobed structure as observed by electron microscopy. The full-length XCGL-1 and the carbohydrate recognition domain (CRD) bind galactose-containing carbohydrates at nanomolar to micromolar affinities. Molecular modeling suggested that galactoside ligands coordinated the binding site calcium ion and interacted with residues around the groove made available by the non-conserved substitution compared to human intelectin-1. Folding conditions for production of recombinant XCGL-1 CRD were also investigated. Our results not only provide new biochemical insights into the function of XCGL-1, but may also provide foundation for further applications of XCGL-1 as glycobiology tools.

Cutaneous lymphoepithelioma-like carcinoma: report of three cases.

BACKGROUND: Cutaneous lymphoepithelioma-like carcinoma (cutaneous LELC) is an extremely rare malignant neoplasm with unclear histogenesis. Its histopathologic features are like those of lymphoepithelioma-like carcinoma occurring in the nasopharynx and in visceral organs especially salivary glands. MATERIAL AND METHOD: The authors reported on one male and two female patients of cutaneous LELC with immunohistochemical and electron microscopic study. All patients were of old age. All cutaneous LELCs in this report occurred on the patient's face, one of each on the eyelid, conjunctiva, and cheek. RESULTS: All resection specimens showed the typical histopathologic features of those of LELC, i.e. well-defined tumor lobules mainly located in the dermis and extending into the panniculus. These tumor lobules typically displayed ill-defined clusters/nests of large epitheloid cells with pale eosinophilic cytoplasm, atypical vesicular nuclei possessing prominent nucleoli and were surrounded by dense lymphoplasmacytic infiltration. Immunocytohistochemically, these epitheloid cells showed epithelial differentiation by the expression of epithelial membrane antigen (EMA), P63, CK5/6 and CAM5.2 but were negative to CK20, CEA and Ebstein-Barr virus (EBV). Squamous cell differentiation by the presence of desmosomes by electron microscopic study was also noted in two patients (case 1 and 3). CONCLUSION: The findings described above indicated that cutaneous LELC was a malignant neoplasm exhibiting squamous cell differentiation.

Enumeration and phenotyping of circulating microvesicles by flow cytometry and nanoparticle tracking analysis: Plasma versus serum.

INTRODUCTION: Microvesicles (MVs) are bioactive, submicron-sized (0.01-1000 nm) membrane vesicles released from various types of cells under normal physiological and pathophysiological conditions. MVs have emerged as important mediators of cell-to-cell communication in a diverse range of normal and pathological processes. MVs have been recognized as potential biomarkers in coagulation, inflammation, and cancer. However, for clinical use, minimizing factors which could affect enumeration and phenotypic characterization of MVs during pre-analytical steps is crucial. In this study, we used flow cytometry and nanoparticle tracking analysis (NTA) to investigate the impact of blood collection using with and without anticoagulant on the number and phenotype of MVs in blood samples. METHODS: Blood from 30 healthy volunteers was collected by venipuncture into 3.2% sodium citrate and clot activator tubes. MV subpopulations and their concentrations were investigated using flow cytometry and NTA. MV morphology was examined by transmission electron microscopy. RESULTS: Results showed that the concentration of MVs was significantly lower in serum than in plasma and that CD41+ MV, CD41+ /CD62P+ MV, CD45+ MV, and CD142+ MV levels from serum were significantly lower than those from plasma, whereas no significant differences in Annexin V (Anx V)+ MV, CD235a+ MV, and CD144+ MV levels were found. Interestingly, serum MVs had a higher proportion of small-sized MVs and lower proportion of large-sized MVs than did plasma MVs. CONCLUSION: Although plasma samples are commonly used, our results suggest that serum can also be used in enumeration of MVs, but care must be taken if coagulation is an aspect of the research.

Ultrastructural Features of Human Liver Specimens from Patients Who Died of Dengue Hemorrhagic Fever.

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA-indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed.

Microparticle and anti-influenza activity in human respiratory secretion.

Respiratory secretions, such as saliva and bronchoalveolar fluid, contain anti-influenza activity. Multiple soluble factors have been described that exert anti-influenza activity and are believed to be responsible for the anti-influenza activity in respiratory secretions. It was previously shown that a bronchial epithelial cell culture could produce exosome-like particles with anti-influenza activity. Whether such extracellular vesicles in respiratory secretions have anti-influenza activity is unknown. Therefore, we characterized bronchoalveolar lavage fluid and found microparticles, which mostly stained positive for epithelial cell markers and both α2,3- and α2,6-linked sialic acid. Microparticles were purified from bronchoalveolar lavage fluid and shown to exhibit anti-influenza activity by a hemagglutination inhibition (HI) assay and a neutralization (NT) assay. In addition, physical binding between influenza virions and microparticles was demonstrated by electron microscopy. These findings indicate that respiratory microparticles containing viral receptors can exert anti-viral activity by probably trapping viral particles. This innate mechanism may play an important role in the defense against respiratory viruses.