Circular Vesicles Research Articles

Experimental study on the effect and mechanism of adipose stem cell-derived exosomes combined with botulinum toxin Aon skin trauma in rats.

Adipose stem cell-derived exosomes (ADSC-EXO) and botulinum toxin type A (BTX-A) individually showed a therapeutic effect on skin wound repair. This study investigated their synergistic effect on promoting skin wound healing in vitro and in vivo and the underlying molecular events. ADSCs were isolated from Sprague-Dawley (SD) rats to obtain ADSC-EXO by ultrafiltration and ultracentrifugation and were confirmed using nanoparticle tracking analysis and transmission electron microscopy. Human skin fibroblasts (HSF) were cultured and treated with or without ADSC-EXO, BTX-A, or their combination. Changes in cell phenotypes and protein expression were analyzed using different in vitro assays, and a rat skin wound model was used to assess their in vivo effects. The isolated ADSC-EXO from primarily cultured ADSCs had a circular vesicle shape with a 30-180 nm diameter. Treatment of HSF with ADSC-EXO and/or BTX-A significantly accelerated HSF migration in vitro and skin wound healing in a rat model. Moreover, ADSC-EXO plus BTX-A treatment dramatically induced VEGFA expression but reduced COL III and COL I levels in vivo. ADSC-EXO and/or BTX-A treatment significantly upregulated TGF-β3 expression on Day 16 after surgery but downregulated TGF-β1 expression, suggesting that ADSC-EXO plus BTX-A promoted skin wound healing and reduced inflammatory cell infiltration. The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-β3/TGF-β1 and COL III/COL I ratio.

Mesenchymal stem cells-derived exosomes ameliorate nucleus pulposus cells apoptosis via delivering miR-142-3p: therapeutic potential for intervertebral disc degenerative diseases

ABSTRACT Intervertebral disc degeneration (IDD) is the main cause of lower back pain (LBP), and puzzles massive individuals worldwide. Mesenchymal stem cells (MSCs) transplantation has been demonstrated to potentially ameliorate IDD progression, while the underlying mechanism has not been fully explained. Interleukin-1β (IL-1β) was used to induce nucleus pulposus cells (NPCs) injury. Bone marrow MSCs-derived exosomes were isolated using the super centrifugation method, and characterized using Transmission electron microscopy (TEM) and western blot. Cell viability was determined by MTT, while apoptosis was measured by Annexin-V staining using flow cytometry. miR-142-3fp and gene expressions were measured by real-time PCR. The protein expressions were determined by western blot. Herein, we found exosomes from bone marrow MSCs are circular vesicles, about 80 nm in diameter, and with robust expression of TSG101 and CD63, but without of Calnexin. MSCs exosomes alleviated NPCs apoptosis by reducing IL-1β-induced inflammatory cytokines secretion and MAPK signaling activation. Additionally, MSCs exosomes inhibited NPCs apoptosis and MAPK signaling by delivering miR-142-3p that targets mixed lineage kinase 3 (MLK3). Overexpression of MLK3 abolished the effects of MSCs exosomes on the inflammatory condition, cell apoptosis, and MAPK signaling activation in NPCs. The results confirmed that bone marrow MSCs-derived exosomes-packaged miR-142-3p alleviates NPCs injury through suppressing MAPK signaling by targeting MLK3. The work highlights the therapeutic effect of MSCs on IDD progression, and bone marrow MSCs exosomes might be apromising therapeutic strategy for IDD.

Follicular extracellular vesicles enhance meiotic resumption of domestic cat vitrified oocytes

Extracellular vesicles (EVs) contain multiple factors that regulate cell and tissue function. However, understanding of their influence on gametes, including communication with the oocyte, remains limited. In the present study, we characterized the proteome of domestic cat (Felis catus) follicular fluid EVs (ffEV). To determine the influence of follicular fluid EVs on gamete cryosurvival and the ability to undergo in vitro maturation, cat oocytes were vitrified using the Cryotop method in the presence or absence of ffEV. Vitrified oocytes were thawed with or without ffEVs, assessed for survival, in vitro cultured for 26 hours and then evaluated for viability and meiotic status. Cat ffEVs had an average size of 129.3 ± 61.7 nm (mean ± SD) and characteristic doughnut shaped circular vesicles in transmission electron microscopy. Proteomic analyses of the ffEVs identified a total of 674 protein groups out of 1,974 proteins, which were classified as being involved in regulation of oxidative phosphorylation, extracellular matrix formation, oocyte meiosis, cholesterol metabolism, glycolysis/gluconeogenesis, and MAPK, PI3K-AKT, HIPPO and calcium signaling pathways. Furthermore, several chaperone proteins associated with the responses to osmotic and thermal stresses were also identified. There were no differences in the oocyte survival among fresh and vitrified oocyte; however, the addition of ffEVs to vitrification and/or thawing media enhanced the ability of frozen-thawed oocytes to resume meiosis. In summary, this study is the first to characterize protein content of cat ffEVs and their potential roles in sustaining meiotic competence of cryopreserved oocytes.

Biological characteristics of Microvesicles Derived from Bone Marrow Mesenchymal Stem Cells and Their Capacities Supporting ex vivo Expansion of Hematopoietic Stem Cells

To explore the biological characteristics of microvesicles(MV) derived from bone marrow mesenchymal stem cells (BM-MSC) and their capability supporting ex vivo expansion of hematopoietic stem cells(HSC). The MV from cultured BM-MSC supernatant were isolated by multi-step differential velocity contrifugation; the morphological characteristics of MV were observed by electron microscopy with negative staining of samples; the protein level in MV was detected by using Micro-BCA method; the surface markers on MV were analyzed by flow cytometry. The peripheral blood HSC(PB-HSC) were isolated after culture and mobilization; the experiment was diveded into 2 group: in MV group, the 10 mg/L MV was given, while in control group, the same volume of PBS was given; the change of PB-HSC count was observed by cell counting; the change of surface markers on PB-HSC was detected dynamically by flow cytometry; the cell colony culture was used to determin the function change of PB-HSC after co-culture with MV. MSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 µg protein could be extracted from every 1×106 MSC. The flow cytometry showed that CD63 and CD44 were positive with a rate of 96.0% and 50.2%, while the HLA-DR, CD34, CD29 and CD73 etc were negative. When being co-cultured with GPBMNC for 2 days, the cell number of MV groups was 1.49±0.15 times of the control group (P>0.05). When being co-cultured for 4 days, the cell number of MV groups was 2.20±0.24 times of the control group(P<0.05). The CD34+ cell number of MV groups was 1.76±0.30 times the control group after culture for 2 day and 1.95±0.20 times after culture for 4 day. The MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.

Shape and Displacement Fluctuations in Soft Vesicles Filled by Active Particles.

We investigate numerically the dynamics of shape and displacement fluctuations of two-dimensional flexible vesicles filled with active particles. At low concentration most of the active particles accumulate at the boundary of the vesicle where positive particle number fluctuations are amplified by trapping, leading to the formation of pinched spots of high density, curvature and pressure. At high concentration the active particles cover the vesicle boundary almost uniformly, resulting in fairly homogeneous pressure and curvature, and nearly circular vesicle shape. The change between polarized and spherical shapes is driven by the number of active particles. The center-of-mass of the vesicle performs a persistent random walk with a long time diffusivity that is strongly enhanced for elongated active particles due to orientational correlations in their direction of propulsive motion. In our model shape-shifting induces directional sensing and the cell spontaneously migrate along the polarization direction.

Characterization and in vitro biocompatibility of catanionic assemblies formed with oppositely charged dicetyl amphiphiles

In this study, cationic dicetyldimethylammonium bromide (DCB) and anionic dicetyl phosphate (DCP) were mixed to form catanionic assemblies in water, and their colloidal morphology, size, charge characteristics, phase behavior, membrane fluidity, and in vitro biocompatibility are comprehensively investigated for the first time. Our results show that the catanionic DCB and DCP mixtures in water are capable of forming circular vesicles and this binary mixed system expresses the miscibility with deviation from the ideal mixing. Compared to the nanoscale DCB-rich vesicles, the DCP-rich vesicles have smaller size, higher negative zeta potential, higher main transition temperature, and better storage stability. The temperature and molecular cooperativity of the main transition phase for the DCP vesicles can be reduced by an addition of DCB. This work demonstrates that the head groups and the relative composition of the two considered dicetyl amphiphiles remarkably affect the phase behavior, membrane rigidity, encapsulation ability and in vitro biocompatibility of DCB/DCP vesicles. The iso-stoichiometric mixed DCB/DCP vesicle, showing low cytotoxicity, long storage time, and high drug loading, is a potential candidate for the drug delivery system.

The influence of porosity and vesicle size on the brittle strength of volcanic rocks and magma

Volcanic rocks and magma display a wide range of porosity and vesicle size, a result of their complex genesis. While the role of porosity is known to exert a fundamental control on strength in the brittle field, less is known as to the influence of vesicle size. To help resolve this issue, here, we lean on a combination of micromechanical (Sammis and Ashby's pore-emanating crack model) and stochastic (rock failure and process analysis code) modelling. The models show, for a homogenous vesicle size, that an increase in porosity (in the form of circular vesicles, from 0 to 40 %) and/or vesicle diameter (from 0.1 to 2.0 mm) results in a dramatic reduction in strength. For example, uniaxial compressive strength can be reduced by about a factor of 5 as porosity is increased from 0 to 40 %. The presence of vesicles locally amplifies the stress within the groundmass and promotes the nucleation of vesicle-emanating microcracks that grow in the direction of the applied macroscopic stress. As strain increases, these microcracks continue to grow and eventually coalesce leading to macroscopic failure. Vesicle clustering, which promotes the overlap and interaction of the tensile stress lobes at the north and south poles of neighbouring vesicles, and the increased ease of microcrack interaction, is encouraged at higher porosity and reduces sample strength. Once a microcrack nucleates at the vesicle wall, larger vesicles impart higher stress intensities at the crack tips, allowing microcracks to propagate at a lower applied macroscopic stress. Larger vesicles also permit a shorter route through the groundmass for the macroscopic shear fracture. This explains the reduction in strength at higher vesicle diameters (at a constant porosity). The modelling highlights that the reduction in strength as porosity or vesicle size increases is nonlinear; the largest reductions are observed at low porosity and small vesicle diameters. In detail, we find that vesicle diameter can play an important role in dictating strength at low porosity but is largely inconsequential above 15 % porosity. Vesicle clustering and stress lobe interaction are implicit at high porosity, regardless of the vesicle diameter. In the case of an inhomogeneous vesicle size, the microcracks grow from the largest vesicles, and brittle strength is closer to that of the largest vesicle end-member. The results of this study highlight the important role of vesicle size, and the complex interplay between porosity and vesicle size, in controlling the brittle strength of volcanic rocks and magma.

Microencapsulation of (deoxythymidine)20–DOTAP complexes in stealth liposomes optimized by Taguchi design

Stealth liposomes encapsulating oligonucleotides are considered as promising non-viral gene delivery carriers; however, general preparation procedures are not capable to encapsulate nucleic acids (NAs) efficiently. In this study, the lyophobic complexes of deoxythymidine20 oligonucleotide (dT20) and DOTAP were used instead of free dT20 for nano-encapsulation process by reverse phase evaporation method. Regarding the various factors that can potentially affect the liposome characteristics, Taguchi design was applied to analyze the simultaneous effects of factors comprising PEG-lipid (%), dT20/total lipid molar ratio, cholesterol (Chol%) and organic-to-aqueous phase ratio (o/w) at three levels. The response variables, hydrodynamic diameter, loading efficiency (LE%) and capacity (LC%), were studied by dynamic light scattering and ethidium bromide exclusion assay, respectively. The optimum condition described by minimum particle size as well as high LE% and LC% was obtained at 5% PEG-lipid, dT20/total lipid of 7, 20% Chol and o/w of 3 with an average size of 84 nm, LE% = 83.4% and LC% = 11.6%. Moreover, stability assessments in presence of heparin sulfate revealed the noticeable resistance, unlike DOTAP/dT20 lipoplexes, to premature release of NA. Transmission electron microscopy confirmed formation of discrete and circular vesicles encapsulating dT20.

Custom‐Made Morphologies of ZnO Nanostructured Films Templated by a Poly(styrene‐block‐ethylene oxide) Diblock Copolymer Obtained by a Sol–Gel Technique

Zinc oxide (ZnO) nanostructured films are synthesized on silicon substrates to form different morphologies that consist of foamlike structures, wormlike aggregates, circular vesicles, and spherical granules. The synthesis involves a sol-gel mechanism coupled with an amphiphilic diblock copolymer poly(styrene-block-ethylene oxide), P(S-b-EO), which acts as a structure-directing template. The ZnO precursor zinc acetate dihydrate (ZAD) is incorporated into the poly(ethylene oxide) block. Different morphologies are obtained by adjusting the weight fractions of the solvents and ZAD. The sizes of the structure in solution for different sol-gels are probed by means of dynamic light scattering. Thin-film samples with ZnO nanostructures are prepared by spin coating and solution casting followed by a calcination step. On the basis of various selected combinations of weight fractions of the ingredients used, a ternary phase diagram is constructed to show the compositional boundaries of the investigated morphologies. The evolution and formation mechanisms of the morphologies are addressed in brief. The surface morphologies of the ZnO nanostructures are studied with SEM. The inner structures of the samples are probed by means of grazing incidence small-angle X-ray scattering to complement the SEM investigations. XRD measurements confirm the crystallization of the ZnO in the wurtzite phase upon calcination of the nanocomposite film in air. The optical properties of ZnO are analyzed by FTIR and UV/Vis spectroscopy.

Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes

All herpesviruses encode a complex of two proteins, referred to as the nuclear egress complex (NEC), which together facilitate the exit of assembled capsids from the nucleus. Previously, we showed that the Kaposi's sarcoma-associated herpesvirus (KSHV) NEC specified by the ORF67 and ORF69 genes when expressed in insect cells using baculoviruses for protein expression forms a complex at the nuclear membrane and remodels these membranes to generate nuclear membrane-derived vesicles. In this study, we have analyzed the functional domains of the KSHV NEC proteins and their interactions. Site-directed mutagenesis of gammaherpesvirus conserved residues revealed functional domains of these two proteins, which in many cases abolish the formation of the NEC and remodeling of nuclear membranes. Small in-frame deletions within ORF67 in all cases result in loss of the ability of the mutant protein to induce cellular membrane proliferation as well as to interact with ORF69. Truncation of the C terminus of ORF67 that resides in the perinuclear space does not impair the functions of ORF67; however, deletion of the transmembrane domain of ORF67 produces a protein that cannot induce membrane proliferation but can still interact with ORF69 in the nucleus and can be tethered to the nuclear membrane by virtue of its interaction with the wild-type-membrane-anchored ORF67. In-frame deletions in ORF69 have varied effects on NEC formation, but all abolish remodeling of nuclear membranes into circular structures. One mutant interacts with ORF67 as well as the wild-type protein but cannot function in membrane curvature and fission events that generate circular vesicles. These studies genetically confirm that ORF67 is required for cellular membrane proliferation and that ORF69 is the factor required to remodel these duplicated membranes into circular-virion-size vesicles. Furthermore, we also investigated the NEC encoded by Epstein-Barr virus (EBV). The EBV complex comprised of BFRF1 and BFLF2 was visualized at the nuclear membrane using autofluorescent protein fusions. BFRF1 is a potent inducer of membrane proliferation; however, BFLF2 cannot remodel these membranes into circular structures. What was evident is the superior remodeling activity of ORF69, which could convert the host membrane proliferations induced by BFRF1 into circular structures.

Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter

Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.

Quantification of vesicle characteristics in some diatreme-filling deposits, and the explosivity levels of magma–water interactions within diatremes

Vesicles within juvenile fragments in mafic pyroclastic deposits contain important information about the state of the magma at the time of fragmentation. There have been few vesicle studies of juvenile pyroclasts from mafic phreatomagmatic deposits, however, and none we can find from maar–diatreme volcanoes. In this paper we document the vesicularity and vesicle-population characteristics of juvenile fragments sampled from non-bedded lithified deposits of the Coombs Hills diatreme complex, part of the Ferrar large igneous province, Antarctica. The diatreme-filling pyroclastic deposits, dominated by lapilli tuffs and tuff breccias, contain typically abundant lithic clasts derived mostly from the enclosing sedimentary sequence, and several types of juvenile clasts ranging from blocky to fluidal or “raggy”.

Microvesicle-mediated RNA Molecule Delivery System Using Monocytes/Macrophages

Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Particle–wall collision statistics in the open circular billiard

In the open circular billiard, particles are placed initially with a uniform distribution in their positions inside a planar circular vesicle. They all have velocities of the same magnitude, whose initial directions are also uniformly distributed. No particle–particle interactions are included, only specular elastic collisions of the particles with the wall of the vesicle. The particles may escape through an aperture with an angle 2 δ . The collisions of the particles with the wall are characterized by the angular position and the angle of incidence. We study the evolution of the system considering the probability distributions of these variables at successive times n the particle reaches the border of the vesicle. These distributions are calculated analytically and measured in numerical simulations. For finite apertures δ < π / 2 , a particular set of initial conditions exists for which the particles are in periodic orbits and never escape the vesicle. This set is of zero measure, but the selection of angular momenta close to these orbits is observed after some collisions, and thus the distributions of probability have a structure formed by peaks. We calculate the marginal distributions up to n = 4 , but for δ > π / 2 a solution is found for arbitrary n . The escape probability as a function of n - 1 decays with an exponent 4 for δ > π / 2 , and evidences for a power law decay are found for lower apertures as well.

Fine structure of normal and degenerating primary afferent boutons associated with characterized spinocervical tract neurons in the cat

Spinocervical tract neurons in the dorsal horn of the cat spinal cord were intracellularly stained with horseradish peroxidase. The neurons came from one intact animal and from animals with dorsal rhizotomies (L3-S2) 3, 5, 10, 28 and 42 days previously. The morphology of terminals associated with spinocervical tract neurons was examined in a combined light and electron microscopical study. Some terminals containing agranular, circular vesicles degenerated as a result of deafferentation; these are therefore the terminals forming monosynaptic inputs to the neurons from primary afferent fibres. Other terminals containing agranular circular vesicles and terminals containing ovoid agranular vesicles survived deafferentation; these boutons therefore do not originate from primary afferent fibres.

Fine structure of spinocervical tract neurones and the synaptic boutons in contact with them

Acute electrophysiological experiments were performed on 7 adult cats and spinocervical tract (SCT) neurones were identified and injected intracellularly with horseradish peroxidase (HRP). At the conclusion of experiments regions of spinal cord were prepared for light and electron microscopy. SCT neurones were studied in thick sections and areas of interest were prepared from the same material for electron microscopy. The general ultrastructural features of SCT neurones were categorised and particular attention was paid to axon terminals contacting SCT dendrites. These terminals were found to be of two varieties: those containing clear circular vesicles and an occasional granular vesicle (63% of total) and those containing elongated agranular vesicles (37% of total).

HERPES ZOSTER OPHTHALMICUS WITH BRIEF REPORT OF FIVE CASES.

Herpes zoster ophthalmicus has been well known for the past third of a century. It is an acute inflammatory affection appearing in the course of the first and second branches of the fifth nerve, and is almost invariably unilateral. It occurs in elevated groups of firm oval or circular vesicles, on an edematous base, at the terminal points of the nerve filaments. The groups of vesicles do not all appear at once and their area is limited to the median line, and to filaments of the branches involved in the inflammation. Herpes zoster ophthalmicus is a neuritis in which the ganglion of Gasser is involved, as has been proven by Do Wecker. Charcot and others have demonstrated that in herpes zoster the ganglion of the posterior root of the spinal nerve is inflamed. The disease is characterized by severe pain, which usually precedes the eruption by a period varying from