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Sample records for vesicle size reduces

  1. Three-Dimensional Culture Reduces Cell Size By Increasing Vesicle Excretion.

    Science.gov (United States)

    Mo, Miaohua; Zhou, Ying; Li, Sen; Wu, Yaojiong

    2018-02-01

    Our previous study has shown that three-dimensional (3D) culture decreases mesenchymal stem cell (MSC) size, leading to enhanced trafficking ability and reduced lung vascular obstructions. However, the underlying mechanisms are unclear. In this study, we proposed that 3D culture reduces MSC size by increasing vesicle excretion. Scanning electron microscope showed that 3D culture markedly increased the amount of membrane-bound vesicles on the cell surface. In consistence, tunable resistive pulse sensing quantifying analysis of vesicles in the culture medium indicated that there were higher levels of vesicles in the 3D culture MSC medium. 3D culture significantly lowered the level of actin polymerization (F-actin), suggestive of lowering actin skeleton tension may facilitate vesicle excretion. Indeed, treatment of MSCs with Cytochalasin D or functional blockade of integrin β1 caused increased vesicle secretion and decreased cell sizes. Thus, our results suggest that 3D culture reduces MSC size by increasing vesicle excretion which is likely mediated by lowering cytoskeleton tension. Stem Cells 2018;36:286-292. © 2017 AlphaMed Press.

  2. Towards traceable size determination of extracellular vesicles

    NARCIS (Netherlands)

    Varga, Zoltán; Yuana, Yuana; Grootemaat, Anita E.; van der Pol, Edwin; Gollwitzer, Christian; Krumrey, Michael; Nieuwland, Rienk

    2014-01-01

    Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. In this manuscript, the size distribution of an

  3. Towards traceable size determination of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Zoltán Varga

    2014-02-01

    Full Text Available Background: Extracellular vesicles (EVs have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. Methods: In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel techniques in the field, such as small-angle X-ray scattering (SAXS and size exclusion chromatography coupled with dynamic light scattering detection. Results: The mode values of the size distributions of the studied erythrocyte EVs reported by the different methods show only small deviations around 130 nm, but there are differences in the widths of the size distributions. Conclusion: SAXS is a promising technique with respect to traceability, as this technique was already applied for traceable size determination of solid nanoparticles in suspension. To reach the traceable measurement of EVs, monodisperse and highly concentrated samples are required.

  4. Detection of extracellular vesicles: size does matter

    NARCIS (Netherlands)

    van der Pol, E.

    2015-01-01

    Cells release small sacks filled with fluid, which are called "extracellular vesicles". The diameter of extracellular vesicles (EV) typically ranges from 30 nm to 1 µm. Because cells release EV into their environment, our body fluids contain numerous EV. Cells release EV to remove waste and to

  5. The BAR Domain Protein PICK1 Controls Vesicle Number and Size in Adrenal Chromaffin Cells

    DEFF Research Database (Denmark)

    da Silva Pinheiro, Paulo César; Jansen, Anna M; de Wit, Heidi

    2014-01-01

    Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6......, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i.......e., reduced quantal size). This was paralleled by a reduction in the mean single-vesicle capacitance, estimated by averaging time-locked capacitance traces. EM confirmed that LDCVs were fewer and of markedly reduced size in the PICK1 KO, demonstrating that all phenotypes can be explained by reductions...

  6. Charged copolypeptide vesicles with controlled size for intracellular drug delivery

    Science.gov (United States)

    Holowka, Eric Peter

    Much focus has been given to the synthesis of polypeptidic based materials due to their unique structural features. These polypeptides commonly are amphiphilic in character that benefit from secondary structural features associated with one of the polymer blocks. These features, such as alpha-helix and beta-sheet conformations, allow for control over nanoscale ordering through self-assembly for use in biological sensors and therapeutic drug delivery. We report the preparation and characterization of charged amphiphilic block copolypeptide vesicle formers using transition metal mediated living ring-opening polymerization of N-carboxyanhydrides (NCAs). The vesicle membranes show fluidic properties suggested by dynamic physical behavior allowing for fine size adjustments using liposomal extrusion methods. This extrusion also allows for a facile mode of encapsulation of biomolecules for drug delivery. Modification of the charged residues has shown vesicle stability under osmotic and thermal stress, in pH buffers, and serum cell media, as well as the ability for lipid interaction and cellular interactions.

  7. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    DEFF Research Database (Denmark)

    Blans, Kristine Ingrid Marie; Hansen, Maria Stenum; Sørensen, Laila V.

    2017-01-01

    Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from...... other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches...... accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles...

  8. Reduction of AP180 and CALM produces defects in synaptic vesicle size and density.

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    Petralia, Ronald S; Wang, Ya-Xian; Indig, Fred E; Bushlin, Ittai; Wu, Fangbai; Mattson, Mark P; Yao, Pamela J

    2013-03-01

    Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180's or CALM's function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.

  9. Size control of giant unilamellar vesicles prepared from inverted emulsion droplets.

    Science.gov (United States)

    Nishimura, Kazuya; Suzuki, Hiroaki; Toyota, Taro; Yomo, Tetsuya

    2012-06-15

    The production of giant lipid vesicles with controlled size and structure will be an important technology in the design of quantitative biological assays in cell-mimetic microcompartments. For establishing size control of giant vesicles, we investigated the vesicle formation process, in which inverted emulsion droplets are transformed into giant unilamellar vesicles (GUVs) when they pass through an oil/water interface. The relationship between the size of the template emulsion and the converted GUVs was studied using inverted emulsion droplets with a narrow size distribution, which were prepared by microfluidics. We successfully found an appropriate centrifugal acceleration condition to obtain GUVs that had a desired size and narrow-enough size distribution with an improved yield so that emulsion droplets can become the template for GUVs. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  10. Quantitative and qualitative analysis of nano-sized vesicles released by dendritic cells and T cells. Towards deciphering the role of extracellular vesicles in immune cell communication

    NARCIS (Netherlands)

    van der Vlist, E.J.|info:eu-repo/dai/nl/314640908

    2013-01-01

    Many cell types release nano-sized vesicles, which can be found in body fluids as well as in cell culture-conditioned medium. These extracellular vesicles (EV) have been identified as vehicles for intercellular communication and are thought to be involved in many (patho)physiological processes. They

  11. A Preferentially Segregated Recycling Vesicle Pool of Limited Size Supports Neurotransmission in Native Central Synapses

    Science.gov (United States)

    Marra, Vincenzo; Burden, Jemima J.; Thorpe, Julian R.; Smith, Ikuko T.; Smith, Spencer L.; Häusser, Michael; Branco, Tiago; Staras, Kevin

    2012-01-01

    Summary At small central synapses, efficient turnover of vesicles is crucial for stimulus-driven transmission, but how the structure of this recycling pool relates to its functional role remains unclear. Here we characterize the organizational principles of functional vesicles at native hippocampal synapses with nanoscale resolution using fluorescent dye labeling and electron microscopy. We show that the recycling pool broadly scales with the magnitude of the total vesicle pool, but its average size is small (∼45 vesicles), highly variable, and regulated by CDK5/calcineurin activity. Spatial analysis demonstrates that recycling vesicles are preferentially arranged near the active zone and this segregation is abolished by actin stabilization, slowing the rate of activity-driven exocytosis. Our approach reveals a similarly biased recycling pool distribution at synapses in visual cortex activated by sensory stimulation in vivo. We suggest that in small native central synapses, efficient release of a limited pool of vesicles relies on their favored spatial positioning within the terminal. PMID:23141069

  12. Single-step isolation of extracellular vesicles by size-exclusion chromatography.

    Science.gov (United States)

    Böing, Anita N; van der Pol, Edwin; Grootemaat, Anita E; Coumans, Frank A W; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. To develop a single-step protocol to isolate vesicles from human body fluids. Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Fractions 9-12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18-20 (32%±2 of total), and protein in fractions 19-21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9-12, with an 8-fold and 70-fold enrichment compared to HDL and protein. SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.

  13. Single-step isolation of extracellular vesicles by size-exclusion chromatography

    Directory of Open Access Journals (Sweden)

    Anita N. Böing

    2014-09-01

    Full Text Available Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim: To develop a single-step protocol to isolate vesicles from human body fluids. Methods: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3. Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL and protein were measured in each fraction. Results: Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively, but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively. HDL was present mainly in fractions 18–20 (32%±2 of total, and protein in fractions 19–21 (36%±2 of total. Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.

  14. Single-step isolation of extracellular vesicles by size-exclusion chromatography

    Science.gov (United States)

    Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. PMID:25279113

  15. Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study

    Science.gov (United States)

    Vestad, Beate; Llorente, Alicia; Neurauter, Axl; Phuyal, Santosh; Kierulf, Bente; Kierulf, Peter; Skotland, Tore; Sandvig, Kirsten; Haug, Kari Bente F.; Øvstebø, Reidun

    2017-01-01

    ABSTRACT Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1–12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised

  16. Size distribution and radial density profile of synaptic vesicles by SAXS and light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Castorph, Simon; Salditt, Tim [Institute for X-ray Physics, Goettingen (Germany); Holt, Matthew; Jahn, Reinhard [Max Plank Institute for Biophysical Chemistry, Goettingen (Germany); Sztucki, Michael [European Synchrotron Radiation Facility, Grenoble (France)

    2008-07-01

    Synaptic vesicles are small membraneous organelles within the nerve terminal, encapsulating neurotransmitters by a lipid bilayer. The transport of the neurotransmitter, the fusion at the plasma membrane, and the release of the stored neurotransmitters into the synaptic cleft are since long know as essential step in nerve conduction of the chemical synapse. A detailed structural view of these molecular mechanisms is still lacking, not withstanding the enormous progress in the field during recent years. From measurements and quantitative fitting of small angle X-ray scattering curves and dynamic light scattering the averaged structural properties of synaptic vesicles can be determined. We present SAXS measurements and fits revealing the width of the size distribution function and details of the radial scattering length profile of synaptic vesicles from rat brain. Representative values for the inner and outer radius and the size polydispersity as well as the density and width of the outer protein layer are obtained.

  17. Experimental Methods to Observe Asymmetric Instability of Intermediate-Reduced-Volume Vesicles in Extensional Flow

    Science.gov (United States)

    Dahl, Joanna; Narsimhan, Vivek; Gouveia, Bernardo; Kumar, Sanjay; Shaqfeh, Eric; Muller, Susan

    2014-11-01

    Vesicles provide an attractive model system to understand the deformation of living cells in response to mechanical forces. These enclosed lipid bilayer membranes are suitable for complementary theoretical and experimental analysis. A recent study (Narsimhan et al., J. Fluid Mech. 750, 144 (2014)) predicted that intermediate-aspect-ratio vesicles break up asymmetrically in extensional flow. Upon infinitesimal perturbation to its shape, the vesicle stretches into an asymmetric dumbbell. In this work, we present preliminary results from cross-slot microfluidic experiments observing this instability. The onset of breakup depends on two non-dimensional parameters: reduced volume (vesicle asphericity) and capillary number (ratio of viscous to bending forces). We will present strategies for accurately measuring these quantities in order to plot a stability diagram. Specifically, we will describe our synthesis of floppy, intermediate-reduced-volume vesicles and our measurement of their bending moduli by analyzing membrane thermal fluctuations. We will discuss coupling particle-image velocimetry (PIV) with cross-slot trapping of vesicles to ensure that breakup occurs at the stagnation point. A preliminary phase diagram for asymmetric breakup will be reported.

  18. Ionic control of the size of the vesicle matrix of beige mouse mast cells.

    Science.gov (United States)

    Curran, M J; Brodwick, M S

    1991-10-01

    Isolated matrices of the giant secretory vesicles of mast cells of the beige mouse were reliably produced by the osmotic lysis of isolated vesicles. These matrices maintained their form, and their sizes were easily measured using Nomarski optics. The size of the matrix depended on the ionic composition of the bathing solution. The physiologically relevant ions, histamine and serotonin, contracted the matrix. Multivalent cations condensed the matrix relative to univalents. Ag+, acid pH (below 5), and basic pH (above 9) expanded the matrix. In the presence of 10 mM histamine, lowering the pH from 9 to 5 contracted the matrix more than can be attributed to the pH-dependent matrix contraction in zero histamine. The nontitratable organic cation, dimethonium, contracts the matrix with little effect of pH in the range of 5-9. These results suggest that histamine acts as a matrix contractor in the divalent form. The dose-response (contraction) relation for histamine was gradual from micromolar to 316 mM (millimolar) histamine. Experiments with mixtures of histamine and sodium show antagonistic effects on the matrix but are inconsistent with either a model where ions compete for identical sites or a parallel model where ions interact with separate independent sites. In vigorous histamine washoff experiments, the half time for vesicle expansion in 10(-4) M pH buffer was approximately 4 s; in isotonic NaCl solution, it was 0.5 s. When 1 M histamine was presented to closely apposed matrices, fusion resulted. The matrix material returned to its initial shape after being mechanically deformed with a glass probe. These results suggest that the matrix size is controlled by its ion exchange properties. The matrix expansion can quantitatively account for the vesicular size increase observed upon exocytosis (as a postfusional event) and the osmotic nonideality of intact vesicles. The mechanical expansion is probably significant in the widening of the exocytotic pore and the dispersal

  19. β-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

    Science.gov (United States)

    Hrynevich, Sviatlana V; Waseem, Tatyana V; Hébert, Audrey; Pellerin, Luc; Fedorovich, Sergei V

    2016-02-01

    The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body β-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by β-hydroxybutyrate. However, in presence of β-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of β-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of β-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by β-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates β-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with

  20. Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

    Directory of Open Access Journals (Sweden)

    R.M. Leão

    2009-01-01

    Full Text Available The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7 or older (p10-12 rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

  1. Single-step isolation of extracellular vesicles by size-exclusion chromatography

    OpenAIRE

    Böing, Anita N.; van der Pol, Edwin; Anita E. Grootemaat; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively.Aim: To develop a single-step protocol to isolate vesicles from human body fluids.Methods: Platelet-free supernatant, derived from platelet...

  2. On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

    Science.gov (United States)

    Deschout, Hendrik; Raemdonck, Koen; Stremersch, Stephan; Maoddi, Pietro; Mernier, Guillaume; Renaud, Philippe; Jiguet, Sébastien; Hendrix, An; Bracke, Marc; van den Broecke, Rudy; Röding, Magnus; Rudemo, Mats; Demeester, Jo; de Smedt, Stefaan C.; Strubbe, Filip; Neyts, Kristiaan; Braeckmans, Kevin

    2014-01-01

    Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics. Here, a microfluidic chip with integrated light sheet illumination is reported, and accurate fluorescence single particle tracking size and concentration measurements of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours are demonstrated.Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics

  3. Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

    Science.gov (United States)

    Marchi-Artzner, V; Gulik-Krzywicki, T; Guedeau-Boudeville, M A; Gosse, C; Sanderson, J M; Dedieu, J C; Lehn, J M

    2001-06-18

    Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured. © 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.

  4. Corticosteroid injections reduce size of rheumatoid nodules

    NARCIS (Netherlands)

    Baan, H.; Baan, H.; Haagsma, C.J.; van de Laar, Mart A F J

    2006-01-01

    Background: Symptomatic rheumatoid nodules are frequently surgically treated. Injection with steroids might be an alternative treatment. Patients and methods: To determine whether injection with triamcinolon acetonide reduces the size of rheumatoid nodules, we randomized twenty patients with

  5. CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

    Science.gov (United States)

    Miller, Sharon E; Mathiasen, Signe; Bright, Nicholas A; Pierre, Fabienne; Kelly, Bernard T; Kladt, Nikolay; Schauss, Astrid; Merrifield, Christien J; Stamou, Dimitrios; Höning, Stefan; Owen, David J

    2015-04-20

    The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration.

    Science.gov (United States)

    Arraud, N; Linares, R; Tan, S; Gounou, C; Pasquet, J-M; Mornet, S; Brisson, A R

    2014-05-01

    Plasma and other body fluids contain membranous extracellular vesicles (EVs), which are considered to derive from activated or apoptotic cells. EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EVs is, however, limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. Our aim was to provide a comprehensive description of EVs from plasma of healthy subjects. Cryo-transmission electron microscopy combined with receptor-specific gold labeling was used to reveal the morphology, size and phenotype of EVs. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EVs. A correlation was performed between conventional flow cytometry and electron microscopy results. We show that platelet-free plasma samples contain spherical EVs, 30 nm to 1 μm in diameter, tubular EVs, 1-5 μm long, and membrane fragments, 1-8 μm large. We show that only a minority of EVs expose the procoagulant lipid phosphatidylserine, in contrast to the classical theory of EV formation. In addition, the concentrations of the main EV sub-populations are determined after sedimentation on EM grids. Finally, we show that conventional flow cytometry, the main method of EV characterization, detects only about 1% of them. This study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations. © 2014 International Society on Thrombosis and Haemostasis.

  7. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

    Science.gov (United States)

    Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

    2016-09-01

    Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

  8. Single-step isolation of extracellular vesicles by size-exclusion chromatography

    NARCIS (Netherlands)

    Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete

  9. A comparison of seminal vesicle size on CT between autosomal dominant polycystic kidney disease (ADPKD) patients and normal subjects

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Ijin; Kim, Seung Hyup; Cho, Jeong Yeon (Dept. of Radiology, Seoul National Univ. College of Medicine, Inst. of Radiation Medicine, Seoul National Univ., Seoul (Korea)), e-mail: kimsh@radcom.snu.ac.kr

    2010-06-15

    Background: Extrarenal manifestations are common in autosomal dominant polycystic kidney disease (ADPKD). Although seminal vesicles can also be involved in patients with ADPKD, little is known about the size differences of the seminal vesicles between ADPKD patients and normal subjects. Purpose: To determine whether the size of seminal vesicles in ADPKD patients is larger than that in normal subjects with the use of three-dimensional (3D) CT. Material and Methods: Using a retrospective case-control study design, we reviewed the findings of 696 male patients with an age range of 20-69 years who underwent contrast enhanced multi-detector computed tomography (MDCT) imaging of the kidney in our institution from August 2007 to July 2008. A total of 68 male patients with ADPKD comprised the study group. Another 68 age-matched non-ADPKD male patients comprised the control group. The size of bilateral seminal vesicles was assessed by measurement of the short dimension on axial, coronal, and sagittal images by the use of a picture archiving and communication system (PACS). Results: The mean width of seminal vesicles in ADPKD patients was 1.70+-0.40 cm (axial images), 1.86+-0.45 cm (coronal), and 1.59+-0.39 cm (sagittal). For control group subjects, the mean width was 1.53+-0.29 cm (axial), 1.68+-0.43 cm (coronal), and 1.48+-0.31 cm (sagittal). The mean size differences between the ADPKD and control groups for the measured widths on axial and coronal images were statistically significant (P=0.01 and P=0.02, respectively). The width as measured on axial images showed a decrease with age in the control group subjects (linear trend, P=0.005), but no significant decrease was noted in ADPKD patients. Conclusion: The seminal vesicles were demonstrated to be larger in ADPKD patients as compared with normal subjects as determined with the use of 3D CT . Keywords: Autosomal dominant polycystic kidney disease (ADPKD), seminal vesicle, computed tomography, CT

  10. Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

    Science.gov (United States)

    Santana, Steven Michael; Antonyak, Marc A.; Cerione, Richard A.; Kirby, Brian J.

    2014-12-01

    Extracellular shed vesicles (ESVs) facilitate a unique mode of cell-cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

  11. Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.

    Science.gov (United States)

    Baumgart, Joel P; Zhou, Zhen-Yu; Hara, Masato; Cook, Daniel C; Hoppa, Michael B; Ryan, Timothy A; Hemmings, Hugh C

    2015-09-22

    Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca(2+) influx without significantly altering the Ca(2+) sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca(2+)]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca(2+) ([Ca(2+)]e). Lowering external Ca(2+) to match the isoflurane-induced reduction in Ca(2+) entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca(2+) entry without significant direct effects on Ca(2+)-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca(2+) influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system.

  12. Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

    Science.gov (United States)

    Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

    2017-08-01

    Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N   G -nitro-l-arginine methyl ester (eNOS inhibitor; P preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease. © 2017 The Authors.

  13. Endothelial Cell-derived Extracellular Vesicles Size-dependently Exert Procoagulant Activity Detected by Thromboelastometry.

    Science.gov (United States)

    Holnthoner, Wolfgang; Bonstingl, Cornelia; Hromada, Carina; Muehleder, Severin; Zipperle, Johannes; Stojkovic, Stefan; Redl, Heinz; Wojta, Johann; Schöchl, Herbert; Grillari, Johannes; Weilner, Sylvia; Schlimp, Christoph J

    2017-06-16

    Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures.

  14. Preparation of large monodisperse vesicles.

    Directory of Open Access Journals (Sweden)

    Ting F Zhu

    Full Text Available Preparation of monodisperse vesicles is important both for research purposes and for practical applications. While the extrusion of vesicles through small pores (approximately 100 nm in diameter results in relatively uniform populations of vesicles, extrusion to larger sizes results in very heterogeneous populations of vesicles. Here we report a simple method for preparing large monodisperse multilamellar vesicles through a combination of extrusion and large-pore dialysis. For example, extrusion of polydisperse vesicles through 5-microm-diameter pores eliminates vesicles larger than 5 microm in diameter. Dialysis of extruded vesicles against 3-microm-pore-size polycarbonate membranes eliminates vesicles smaller than 3 microm in diameter, leaving behind a population of monodisperse vesicles with a mean diameter of approximately 4 microm. The simplicity of this method makes it an effective tool for laboratory vesicle preparation with potential applications in preparing large monodisperse liposomes for drug delivery.

  15. Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements.

    Science.gov (United States)

    Kumar, Abhijeet; Gilson, Laurent; Henrich, Franziska; Dahl, Verena; Kleinen, Jochen; Gambaryan-Roisman, Tatiana; Venzmer, Joachim

    2016-07-01

    Understanding the mechanism of intact vesicle deposition on solid surfaces is important for effective utilization of vesicles as active ingredient carriers in applications such as drug delivery and fabric softening. In this study, the deposition of large (davg=12μm) and small (davg=0.27μm) cationic vesicles of ditallowethylester dimethylammonium chloride (DEEDMAC) on smooth and rough anionic cellulose fibers is investigated. The deposition process is studied quantitatively using streaming potential measurements and spectrophotometric determination of DEEDMAC concentrations. Natural and regenerated cellulose fibers, namely cotton and viscose, having rough and smooth surfaces, respectively, are used as adsorbents. Equilibrium deposition data and profiles of substrate streaming potential variation with deposition are used to gain insights into the fate of vesicles upon deposition and the deposition mechanism. Intact deposition of DEEDMAC vesicles is ascertained based on streaming potential variation with deposition in the form of characteristic saturating profiles which symbolize particle-like deposition. The same is also confirmed by confocal fluorescence microscopy. Substrate roughness is found to considerably influence the deposition mechanism which, in a novel application of electrokinetic methods, is elucidated via streaming potential measurements. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    Science.gov (United States)

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity.

  17. Dynamic properties of the alkaline vesicle population at hippocampal synapses.

    Directory of Open Access Journals (Sweden)

    Mareike Röther

    Full Text Available In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval.

  18. Elevated Abundance, Size, and MicroRNA Content of Plasma Extracellular Vesicles in Viremic HIV-1+ Patients: Correlations With Known Markers of Disease Progression.

    Science.gov (United States)

    Hubert, Audrey; Subra, Caroline; Jenabian, Mohammad-Ali; Tremblay Labrecque, Pierre-François; Tremblay, Cécile; Laffont, Benoit; Provost, Patrick; Routy, Jean-Pierre; Gilbert, Caroline

    2015-11-01

    Because of factors only partly understood, the generalized elevated immune activation and inflammation characterizing HIV-1-infected patients are corrected incompletely with antiretroviral therapy (ART). Extracellular vesicles (EVs) including exosomes and microvesicles released by several cell types may contribute to immune activation and dysfunction. EV size, abundance, and content appear to differ according to infection phase, disease progression, and ART. We examined whether the size of EVs and the abundance of exosomes in plasma are associated with cell and tissue activation as well as with viral production. Acetylcholinesterase-bearing (AChE+) exosomes in plasma were quantified using an AChE assay. EV size was analyzed using dynamic light scattering. Proteins and microRNAs present in EVs were detected by Western blot and real-time polymerase chain reaction, respectively. Exosomes were found more abundant in the plasma of ART-naive patients. EV size was larger in ART-naive than in ART-suppressed patients, elite controllers, or healthy control subjects. Both exosome abundance and EV sizes were inversely correlated with CD4/CD8 T-cell ratio and neutrophil, platelet, and CD4 T-cell counts and positively correlated with CD8 T-cell counts. A negative correlation was found between CD4 T-cell nadir and exosome abundance, but not EV size. Levels of miR-155 and miR-223 but not miR-92 were strongly correlated negatively with EV abundance and size in ART-naive patients. Monitoring of circulating EVs and EV-borne microRNA is possible and may provide new insight into HIV-1 pathogenesis, disease progression, and the associated inflammatory state, as well as the efficacy of ART and the treatments intended to reduce immune activation.

  19. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  20. Inventions on reducing keyboard size: A TRIZ based analysis

    OpenAIRE

    Mishra, Umakant

    2013-01-01

    A conventional computer keyboard consists of as many as 101 keys. The keyboard has several sections, such as text entry section, navigation section, and numeric keypad etc. and each having several keys on the keyboard. The size of the keyboard is a major inconvenience for portable computers, as they cannot be carried easily. Thus there are certain circumstances which compels to reduce the size of a keyboard. Reducing the size of a keyboard leads to several problems. A reduced size keyboard ma...

  1. Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

    Science.gov (United States)

    Saito, Akira C; Ogura, Toshihiko; Fujiwara, Kei; Murata, Satoshi; Nomura, Shin-ichiro M

    2014-01-01

    Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell-GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

  2. Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

    Directory of Open Access Journals (Sweden)

    Akira C Saito

    Full Text Available Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC field to induce a linear cell-GUV alignment, and then a direct current (DC pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

  3. Addition of thrombin reduces the recovery of extracellular vesicles from blood plasma

    Science.gov (United States)

    Arakelyan, Anush; Fitzgerald, Wendy; Vagida, Murad; Vasilieva, Elena; Grivel, Jean-Charles

    2016-01-01

    Extracellular vesicles (EVs) are widely studied as a system of intercellular communication, as markers of various diseases, as well as a vehicle for delivery of various bioactive molecules to various cells. Investigation of EVs’ structure and function requires their isolation and precise quantification. However, in the current literature, there are significant discrepancies in the estimated numbers of EVs in different body fluids. In part, this discrepancy is due to the difference in EVs isolation protocols used by different investigators. A common protocol that includes ExoQuick™ is often used to isolate EVs from body fluids and culture medium. Here, we show that in the case of isolation of EVs from blood, thrombin should be omitted from the protocol as clots formed due to the thrombin-triggered coagulation may entrap many EVs thus leading to the underestimation of their numbers. PMID:28936260

  4. Addition of thrombin reduces the recovery of extracellular vesicles from blood plasma

    Directory of Open Access Journals (Sweden)

    Anush Arakelyan

    2016-10-01

    Full Text Available Extracellular vesicles (EVs are widely studied as a system of intercellular communication, as markers of various diseases, as well as a vehicle for delivery of various bioactive molecules to various cells. Investigation of EVs’ structure and function requires their isolation and precise quantification. However, in the current literature, there are significant discrepancies in the estimated numbers of EVs in different body fluids. In part, this discrepancy is due to the difference in EVs isolation protocols used by different investigators. A common protocol that includes ExoQuick ™ is often used to isolate EVs from body fluids and culture medium. Here, we show that in the case of isolation of EVs from blood, thrombin should be omitted from the protocol as clots formed due to the thrombin-triggered coagulation may entrap many EVs thus leading to the underestimation of their numbers.

  5. Candida albicans Modifies the Protein Composition and Size Distribution of THP-1 Macrophage-Derived Extracellular Vesicles.

    Science.gov (United States)

    Reales-Calderón, Jose Antonio; Vaz, Catarina; Monteoliva, Lucía; Molero, Gloria; Gil, Concha

    2017-01-06

    The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, noncoding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus we have undertaken the first quantitative proteomic analysis on the protein composition of THP-1 macrophage-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP-1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP-1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP-1 nondifferenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.

  6. Vesicle Photonics

    Science.gov (United States)

    Vasdekis, A. E.; Scott, E. A.; Roke, S.; Hubbell, J. A.; Psaltis, D.

    2013-07-01

    Amphiphiles, under appropriate conditions, can self-assemble into nanoscale thin membrane vessels (vesicles) that encapsulate and hence protect and transport molecular payloads. Vesicles assemble naturally within cells but can also be artificially synthesized. In this article, we review the mechanisms and applications of light-field interactions with vesicles. By being associated with light-emitting entities (e.g., dyes, fluorescent proteins, or quantum dots), vesicles can act as imaging agents in addition to cargo carriers. Vesicles can also be optically probed on the basis of their nonlinear response, typically from the vesicle membrane. Light fields can be employed to transport vesicles by using optical tweezers (photon momentum) or can directly perturb the stability of vesicles and hence trigger the delivery of the encapsulated payload (photon energy). We conclude with emerging vesicle applications in biology and photochemical microreactors.

  7. Size and CT density of iodine-containing ethosomal vesicles obtained by membrane extrusion: potential for use as CT contrast agents.

    Science.gov (United States)

    Na, Bomin; Choi, Byoung Wook; Kim, Bumsang

    2013-11-01

    Computed tomography (CT) is the primary non-invasive imaging technique used for most patients with suspected liver disease. In order to improve liver-specific imaging properties and prevent toxic effects in patients with compromised renal function, we investigated the encapsulation of iodine within ethosomal vesicles. As a first step in the development of novel contrast agents using ethosomes for CT imaging applications, iodine was entrapped within ethosomes and iodine-containing ethosomes of the desired size were obtained by extrusion using a polycarbonate membrane with a defined pore size. Ethosomes containing iodine showed a relatively high CT density, which decreased when they were extruded, due to the rupture and re-formation of the lipid bilayer of the ethosome. However, when a solution with a high iodine concentration was used as a dispersion media during the extrusion process, the decrease in CT density could be prevented. In addition, ethosomes containing iodine were taken up efficiently by macrophages, which are abundant in the liver, and these ethosomes exhibited no cellular toxicity. These results demonstrate that iodine could be entrapped within ethosomal vesicles, giving the ethosomes a relatively high CT density, and that the extrusion technique used in this study could conveniently and reproducibly produce ethosomal vesicles with a desired size. Therefore, ethosomes containing iodine, as prepared in this study, have potential as contrast agents with applications in CT imaging. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Experimental Analysis of Reduced-Sized Coplanar Waveguide Transmission Lines

    Science.gov (United States)

    Ponchak, George E.

    2002-01-01

    An experimental investigation of the use of capacitive loading of coplanar waveguides to reduce their line length and, thus the size, of monolithic microwave integrated circuits is presented. The reduced sized coplanar waveguides are compared to unloaded transmission lines and to lumped element transmission line segments. The phase bandwidth, defined by 2 percent error in S(sub 21), and the return loss bandwidth, defined by a return loss greater than 15 dB, of coplanar waveguides reduced from 0 to 90 percent are compared, and the insertion loss as a function of the size reduction is presented.

  9. Outer membrane vesicles harboring modified lipid A moiety augment the efficacy of an influenza vaccine exhibiting reduced endotoxicity in a mouse model.

    Science.gov (United States)

    Lee, Tae-Young; Kim, Chang-Ung; Bae, Eun-Hye; Seo, Sang-Hwan; Jeong, Dae Gwin; Yoon, Sun-Woo; Chang, Kyu-Tae; Kim, Young Sang; Kim, Sang-Hyun; Kim, Doo-Jin

    2017-01-23

    Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103+ dendritic cells in the mediastinal lymph nodes was significantly increased after fmOMV co-administration. Notably, the mice co-immunized with fmOMV showed a significantly higher protection rate against challenge with a lethal dose of homologous or heterologous influenza viruses without adverse effects. These results show the potential of fmOMV as an effective mucosal adjuvant for intranasal vaccines. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Elastic energy of polyhedral bilayer vesicles.

    Science.gov (United States)

    Haselwandter, Christoph A; Phillips, Rob

    2011-06-01

    In recent experiments [M. Dubois, B. Demé, T. Gulik-Krzywicki, J.-C. Dedieu, C. Vautrin, S. Désert, E. Perez, and T. Zemb, Nature (London) 411, 672 (2001)] the spontaneous formation of hollow bilayer vesicles with polyhedral symmetry has been observed. On the basis of the experimental phenomenology it was suggested [M. Dubois, V. Lizunov, A. Meister, T. Gulik-Krzywicki, J. M. Verbavatz, E. Perez, J. Zimmerberg, and T. Zemb, Proc. Natl. Acad. Sci. USA 101, 15082 (2004)] that the mechanism for the formation of bilayer polyhedra is minimization of elastic bending energy. Motivated by these experiments, we study the elastic bending energy of polyhedral bilayer vesicles. In agreement with experiments, and provided that excess amphiphiles exhibiting spontaneous curvature are present in sufficient quantity, we find that polyhedral bilayer vesicles can indeed be energetically favorable compared to spherical bilayer vesicles. Consistent with experimental observations we also find that the bending energy associated with the vertices of bilayer polyhedra can be locally reduced through the formation of pores. However, the stabilization of polyhedral bilayer vesicles over spherical bilayer vesicles relies crucially on molecular segregation of excess amphiphiles along the ridges rather than the vertices of bilayer polyhedra. Furthermore, our analysis implies that, contrary to what has been suggested on the basis of experiments, the icosahedron does not minimize elastic bending energy among arbitrary polyhedral shapes and sizes. Instead, we find that, for large polyhedron sizes, the snub dodecahedron and the snub cube both have lower total bending energies than the icosahedron.

  11. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

  12. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties

    NARCIS (Netherlands)

    Nordin, Joel Z.; Lee, Yi; Vader, Pieter; Mäger, Imre; Johansson, Henrik J.; Heusermann, Wolf; Wiklander, Oscar P B; Hällbrink, Mattias; Seow, Yiqi; Bultema, Jarred J.; Gilthorpe, Jonathan; Davies, Tim; Fairchild, Paul J.; Gbrielsson, Susanne; Meisner-Kober, Nicole C.; Lehtiö, Janne; Smith, C. I Edvard; Wood, Matthew J A; Andaloussi, Samir E L

    2015-01-01

    Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and

  13. Inter-laboratory comparison on the size and stability of monodisperse and bimodal synthetic reference particles for standardization of extracellular vesicle measurements

    Science.gov (United States)

    Nicolet, Anaïs; Meli, Felix; van der Pol, Edwin; Yuana, Yuana; Gollwitzer, Christian; Krumrey, Michael; Cizmar, Petr; Buhr, Egbert; Pétry, Jasmine; Sebaihi, Noham; de Boeck, Bert; Fokkema, Vincent; Bergmans, Rob; Nieuwland, Rienk

    2016-03-01

    In future, measurements of extracellular vesicles in body fluids could become a standard diagnostic tool in medicine. For this purpose, reliable and traceable methods, which can be easily applied in hospitals, have to be established. Within the European Metrological Research Project (EMRP) ‘Metrological characterization of micro-vesicles from body fluids as non-invasive diagnostic biomarkers’ (www.metves.eu), various nanoparticle reference materials were developed and characterized. We present results of an international comparison among four national metrology institutes and a university hospital. The size distributions of five monodisperse and two bimodal spherical particle samples with diameters ranging from 50 nm to 315 nm made out of silica and polystyrene were compared. Furthermore, the stability of the samples was verified over a period of 18 months. While monodisperse reference particle samples above a certain size level lead to good agreements of the size measurements among the different methods, small and bimodal samples show the limitations of current ‘clinical’ methods. All samples proved to be stable within the uncertainty of the applied methods.

  14. Aceclofenac encapsulated ethanolic nano-vesicles for effective treatment of osteoarthritis

    National Research Council Canada - National Science Library

    Kaur, Arvinder; Jain, Sunil K; Pandey, Ravi S

    2012-01-01

    .... Ethanolic nano-vesicles were prepared by solvent dispersion method. Vesicles were characterized for vesicular size, surface morphology, size and size distribution, zeta potential, entrapment efficiency...

  15. Reduced genome size of Helicobacter pylori originating from East Asia.

    Science.gov (United States)

    Dong, Quan-Jiang; Wang, Li-Li; Tian, Zi-Bing; Yu, Xin-Jun; Jia, Sheng-Jiao; Xuan, Shi-Ying

    2014-05-21

    Helicobacter pylori (H. pylori), a major pathogen colonizing the human stomach, shows great genetic variation. Comparative analysis of strains from different H. pylori populations revealed that the genome size of strains from East Asia decreased to 1.60 Mbp, which is significantly smaller than that from Europe or Africa. In parallel with the genome reduction, the number of protein coding genes was decreased, and the guanine-cytosine content was lowered to 38.9%. Elimination of non-essential genes by mutations is likely to be a major cause of the genome reduction. Bacteria with a small genome cost less energy. Thus, H. pylori strains from East Asia may have proliferation and growth advantages over those from Western countries. This could result in enhanced capacity of bacterial spreading. Therefore, the reduced genome size potentially contributes to the high prevalence of H. pylori in East Asia.

  16. Red maca (Lepidium meyenii reduced prostate size in rats

    Directory of Open Access Journals (Sweden)

    Rubio Julio

    2005-01-01

    Full Text Available Abstract Background Epidemiological studies have found that consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer. This effect seems to be due to aromatic glucosinolate content. Glucosinolates are known for have both antiproliferative and proapoptotic actions. Maca is a cruciferous cultivated in the highlands of Peru. The absolute content of glucosinolates in Maca hypocotyls is relatively higher than that reported in other cruciferous crops. Therefore, Maca may have proapoptotic and anti-proliferative effects in the prostate. Methods Male rats treated with or without aqueous extracts of three ecotypes of Maca (Yellow, Black and Red were analyzed to determine the effect on ventral prostate weight, epithelial height and duct luminal area. Effects on serum testosterone (T and estradiol (E2 levels were also assessed. Besides, the effect of Red Maca on prostate was analyzed in rats treated with testosterone enanthate (TE. Results Red Maca but neither Yellow nor Black Maca reduced significantly ventral prostate size in rats. Serum T or E2 levels were not affected by any of the ecotypes of Maca assessed. Red Maca also prevented the prostate weight increase induced by TE treatment. Red Maca administered for 42 days reduced ventral prostatic epithelial height. TE increased ventral prostatic epithelial height and duct luminal area. These increases by TE were reduced after treatment with Red Maca for 42 days. Histology pictures in rats treated with Red Maca plus TE were similar to controls. Phytochemical screening showed that aqueous extract of Red Maca has alkaloids, steroids, tannins, saponins, and cardiotonic glycosides. The IR spectra of the three ecotypes of Maca in 3800-650 cm (-1 region had 7 peaks representing 7 functional chemical groups. Highest peak values were observed for Red Maca, intermediate values for Yellow Maca and low values for Black Maca. These functional groups correspond among others to benzyl

  17. Red maca (Lepidium meyenii) reduced prostate size in rats

    Science.gov (United States)

    Gonzales, Gustavo F; Miranda, Sara; Nieto, Jessica; Fernández, Gilma; Yucra, Sandra; Rubio, Julio; Yi, Pedro; Gasco, Manuel

    2005-01-01

    Background Epidemiological studies have found that consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer. This effect seems to be due to aromatic glucosinolate content. Glucosinolates are known for have both antiproliferative and proapoptotic actions. Maca is a cruciferous cultivated in the highlands of Peru. The absolute content of glucosinolates in Maca hypocotyls is relatively higher than that reported in other cruciferous crops. Therefore, Maca may have proapoptotic and anti-proliferative effects in the prostate. Methods Male rats treated with or without aqueous extracts of three ecotypes of Maca (Yellow, Black and Red) were analyzed to determine the effect on ventral prostate weight, epithelial height and duct luminal area. Effects on serum testosterone (T) and estradiol (E2) levels were also assessed. Besides, the effect of Red Maca on prostate was analyzed in rats treated with testosterone enanthate (TE). Results Red Maca but neither Yellow nor Black Maca reduced significantly ventral prostate size in rats. Serum T or E2 levels were not affected by any of the ecotypes of Maca assessed. Red Maca also prevented the prostate weight increase induced by TE treatment. Red Maca administered for 42 days reduced ventral prostatic epithelial height. TE increased ventral prostatic epithelial height and duct luminal area. These increases by TE were reduced after treatment with Red Maca for 42 days. Histology pictures in rats treated with Red Maca plus TE were similar to controls. Phytochemical screening showed that aqueous extract of Red Maca has alkaloids, steroids, tannins, saponins, and cardiotonic glycosides. The IR spectra of the three ecotypes of Maca in 3800-650 cm (-1) region had 7 peaks representing 7 functional chemical groups. Highest peak values were observed for Red Maca, intermediate values for Yellow Maca and low values for Black Maca. These functional groups correspond among others to benzyl glucosinolate. Conclusions

  18. Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry.

    Directory of Open Access Journals (Sweden)

    Carla Pérez-Cruz

    Full Text Available Membrane vesicles (MVs produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs and the recently described double-bilayer vesicles (O-IMVs, other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications.

  19. Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.

    Science.gov (United States)

    Weilner, Sylvia; Keider, Verena; Winter, Melanie; Harreither, Eva; Salzer, Benjamin; Weiss, Florian; Schraml, Elisabeth; Messner, Paul; Pietschmann, Peter; Hildner, Florian; Gabriel, Christian; Redl, Heinz; Grillari-Voglauer, Regina; Grillari, Johannes

    2016-01-01

    Aging results in a decline of physiological functions and in reduced repair capacities, in part due to impaired regenerative power of stem cells, influenced by the systemic environment. In particular osteogenic differentiation capacity (ODC) of mesenchymal stem cells (MSCs) has been shown to decrease with age, thereby contributing to reduced bone formation and an increased fracture risk. Searching for systemic factors that might contribute to this age related decline of regenerative capacity led us to investigate plasma-derived extracellular vesicles (EVs). EVs of the elderly were found to inhibit osteogenesis compared to those of young individuals. By analyzing the differences in the vesicular content Galectin-3 was shown to be reduced in elderly-derived vesicles. While overexpression of Galectin-3 resulted in an enhanced ODC of MSCs, siRNA against Galectin-3 reduced osteogenesis. Modulation of intravesicular Galectin-3 levels correlated with an altered osteo-inductive potential indicating that vesicular Galectin-3 contributes to the biological response of MSCs to EVs. By site-directed mutagenesis we identified a phosphorylation-site on Galectin-3 mediating this effect. Finally, we showed that cell penetrating peptides comprising this phosphorylation-site are sufficient to increase ODC in MSCs. Therefore, we suggest that decrease of Galectin-3 in the plasma of elderly contributes to the age-related loss of ODC.

  20. Vesicles and vesicle gels - structure and dynamics of formation

    CERN Document Server

    Gradzielski, M

    2003-01-01

    Vesicles constitute an interesting morphology formed by self-aggregating amphiphilic molecules. They exhibit a rich structural variety and are of interest both from a fundamental point of view (for studying closed bilayer systems) and from a practical point of view (whenever one is interested in the encapsulation of active molecules). In many circumstances vesicular structures have to be formed by external forces, but of great interest are amphiphilic systems, where they form spontaneously. Here the question arises of whether this means that they are also thermodynamically stable structures, which at least in some systems appears to be the case. If such vesicles are well defined in size, it is possible to pack them densely and thereby form vesicle gels that possess highly elastic properties even for relatively low volume fractions of amphiphile. Conditions for the formation and the microstructure of such vesicle gels have been studied in some detail for the case of unilamellar vesicles. Another important and ...

  1. Trichomonas vaginalis exosome-like vesicles modify the cytokine profile and reduce inflammation in parasite-infected mice.

    Science.gov (United States)

    Olmos-Ortiz, L M; Barajas-Mendiola, M A; Barrios-Rodiles, M; Castellano, L E; Arias-Negrete, S; Avila, E E; Cuéllar-Mata, P

    2017-06-01

    Trichomonas vaginalis (Tv) is a flagellated parasite commonly spread through sexual transmission. This protozoan initiates a severe inflammatory process, inducing nitric oxide, interleukin-6 (IL-6), IL-8, IL-10, IL-17 and IL-22 production by host immune cells. The parasites elicit these responses by releasing surface lipophosphoglycan, small extracellular vesicles (exosomes) and other factors. Tv exosomes are similar to mammalian exosomes and have been implicated in the modulation of IL-8 secretion by epithelial cells. Here, we report that exosome-like vesicles from T. vaginalis (Tv-ELVs) induced a more than 15-fold increase in IL-10 expression in RAW264.7 macrophages but only a two fold increase in IL-6 and TNF-α expression levels measured by RT-PCR. Because Tv-ELVs modulated the macrophage response, we also explored the effect of Tv-ELVs in a murine model of infection. Pretreatment with Tv-ELVs significantly increased IL-10 production as measured in vaginal washes by days 8 and 16 post-infection. Remarkably, Tv-ELVs-pretreated mice exhibited a decrease in IL-17 production and a significant decrease in vulvar inflammation. In addition, IL-6 and IL-13 were decreased during infection. Our results suggest that Tv-ELVs have an immunomodulatory role on the cytokine profile induced by the parasite and promote a decrease in the inflammatory process in mice infected with T. vaginalis. © 2017 John Wiley & Sons Ltd.

  2. Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum.

    Science.gov (United States)

    Matos Baltazar, Ludmila; Nakayasu, Ernesto S; Sobreira, Tiago J P; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D

    2016-01-01

    Histoplasma capsulatum produces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment of H. capsulatum cells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bind H. capsulatum heat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion. IMPORTANCE Diverse fungal species release extracellular vesicles, indicating that this is a common pathway for the delivery of molecules to the extracellular space. However, there has

  3. The readily releasable pool of synaptic vesicles.

    Science.gov (United States)

    Kaeser, Pascal S; Regehr, Wade G

    2017-04-01

    Each presynaptic bouton is densely packed with many vesicles, only a small fraction of which are available for immediate release. These vesicles constitute the readily releasable pool (RRP). The RRP size, and the probability of release of each vesicle within the RRP, together determine synaptic strength. Here, we discuss complications and recent advances in determining the size of the physiologically relevant RRP. We consider molecular mechanisms to generate and regulate the RRP, and discuss the relationship between vesicle docking and the RRP. We conclude that many RRP vesicles are docked, that some docked vesicles may not be part of the RRP, and that undocked vesicles can contribute to the RRP by rapid recruitment to unoccupied, molecularly activated ready-to-release sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Tunable Reduced Size Planar Folded Slot Antenna Utilizing Varactor Diodes

    Science.gov (United States)

    Scardelletti, Maximilian C.; Ponchak, George E.; Jordan, Jennifer L.; Jastram, Nathan; Mahaffey, Joshua V.

    2010-01-01

    A tunable folded slot antenna that utilizes varactor diodes is presented. The antenna is fabricated on Rogers 6006 Duriod with a dielectric constant and thickness of 6.15 and 635 m, respectively. A copper cladding layer of 17 m defines the antenna on the top side (no ground on backside). The antenna is fed with a CPW 50 (Omega) feed line, has a center frequency of 3 GHz, and incorporates Micrometrics microwave hyper-abrupt 500MHV varactors to tune the resonant frequency. The varactors have a capacitance range of 2.52 pF at 0 V to 0.4 pF at 20 V; they are placed across the radiating slot of the antenna. The tunable 10 dB bandwidth of the 3 GHz antenna is 150 MHz. The varactors also reduce the size of the antenna by 30% by capacitively loading the resonating slot line. At the center frequency, 3 GHz, the antenna has a measured return loss of 44 dB and a gain of 1.6 dBi. Full-wave electromagnetic simulations using HFSS are presented that validate the measured data. Index Terms capacitive loading, Duriod, folded slot antenna, varactor.

  5. Changes in numbers and size of synaptic vesicles of cortical neurons induced by exposure to 835 MHz radiofrequency-electromagnetic field

    Science.gov (United States)

    Yu, Da-Hyeon; Kweon, Hee-Seok; Huh, Yang Hoon

    2017-01-01

    We studied the effects of radiofrequency electromagnetic fields (RF-EMFs) exposure on neuronal functions of mice. Particularly, we focused on RF-EMF effects on synaptic vesicles (SVs), which store neurotransmitters at axon terminals or synaptic boutons. C57 BL/6 mice were exposed to 835 MHz RF-EMF (4.0 W/kg SAR, for 5 h daily) and alterations in SVs at presynaptic terminals in the cerebral cortex were determined. Ultrastructure of randomly selected cortical neurons was observed using typical electron microscopy and bio-high voltage electron microscopy (Bio-HVEM) methods, which enable the estimation of the numbers and size of SVs. The density of the SVs (number /10 μm2 or 40 μm3) was significantly decreased in the presynaptic boutons of cortical neurons after RF-EMF exposure. Furthermore, qPCR and immunoblotting analyses revealed that the expression of synapsins I/II (Syns I/II) genes and proteins were significantly decreased in the cortical neurons of RF-EMF exposed mice. The present study suggested that alteration of SVs and Syn levels may result in alterations of neurotransmitters in the cerebral cortex following RF-EMF exposure. PMID:29045446

  6. Immunotherapeutic Potential of Extracellular Vesicles

    OpenAIRE

    Zhang, Bin; Yin, Yijun; Lai, Ruenn Chai; Lim, Sai Kiang

    2014-01-01

    Extracellular vesicle or EV is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes, the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized...

  7. PDGF-BB Carried by Endothelial Cell-derived Extracellular Vesicles Reduces Vascular Smooth Muscle Cell Apoptosis in Diabetes.

    Science.gov (United States)

    Togliatto, Gabriele; Dentelli, Patrizia; Rosso, Arturo; Lombardo, Giusy; Gili, Maddalena; Gallo, Sara; Gai, Chiara; Solini, Anna; Camussi, Giovanni; Brizzi, Maria Felice

    2018-01-31

    Endothelial cell-derived extracellular vesicles (CD31EVs) are a new entity for therapeutic/prognostic purposes. The roles of CD31EVs as mediators of smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2D) is investigated herein.We demonstrated that, unlike non-diabetic, diabetic serum-derived-EVs (D-CD31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycaemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between anti-apoptotic/pro-apoptotic signals: increase of bcl-2 and decrease of bak/bax. D-CD31EV cargo analysis demonstrated that D-CD31EVs are enriched in membrane-bound-platelet-derived-growth-factor-BB (mbPDGF-BB). Thus, we postulated that mbPDGF-BB transfer by D-CD31EVs could account for VSMC resistance to apoptosis. By depleting CD31EVs of PDGF-BB or blocking the PDGF-BB-receptorβ on VSMCs, we demonstrated that mbPDGF-BB contributes to D-CD31EV-mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of PDGF-BB-mediated miR-296-5p expression. In fact, while PDGF-BB-treatment recapitulated D-CD31EV-mediated anti-apoptotic program and VSMC resistance to apoptosis, PDGF-BB-depleted CD31EVs failed. D-CD31EVs also increased VSMC migration and recruitment to neovessels, by means of PDGF-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of T2D individuals, express low bak/bax and high bcl-2 and miR-296-5p levels.This study identifies the mbPDGF-BB in D-CD31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis. © 2018 by the American Diabetes Association.

  8. Reversibly formed bilayer vesicles: Energetics and polydispersity

    DEFF Research Database (Denmark)

    Bergstöm, M.

    1997-01-01

    orders of magnitude larger than where the local free energy minima of the equilibrium vesicle actually occur. Moreover, according to our analysis, the relative width of a vesicle size distribution, sigma(R)/R-max, is generally at full equilibrium equal to 0.283, independently of the energetic vesicle....... and a statistical-mechanical factor that accounts for the fluctuations in composition, chain packing density and shape. We demonstrate that the free energy required to form a spherical vesicle is made up of two main contributions: the (size-independent) work of bending the constituent monolayers and the work...

  9. Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease.

    Science.gov (United States)

    Hook, Vivian; Toneff, Thomas; Bogyo, Matthew; Greenbaum, Doron; Medzihradszky, Katalin F; Neveu, John; Lane, William; Hook, Gregory; Reisine, Terry

    2005-09-01

    The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.

  10. Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

    Energy Technology Data Exchange (ETDEWEB)

    Baltazar, Ludmila M.; Nakayasu, Ernesto S.; Sobreira, Tiago; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.

    2016-03-30

    ABSTRACT

    Histoplasma capsulatumproduces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment ofH. capsulatumcells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bindH. capsulatumheat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion.

    IMPORTANCEDiverse fungal species release extracellular vesicles, indicating that this is a

  11. High Speed Gear Sized and Configured to Reduce Windage Loss

    Science.gov (United States)

    Kunz, Robert F. (Inventor); Medvitz, Richard B. (Inventor); Hill, Matthew John (Inventor)

    2013-01-01

    A gear and drive system utilizing the gear include teeth. Each of the teeth has a first side and a second side opposite the first side that extends from a body of the gear. For each tooth of the gear, a first extended portion is attached to the first side of the tooth to divert flow of fluid adjacent to the body of the gear to reduce windage losses that occur when the gear rotates. The gear may be utilized in drive systems that may have high rotational speeds, such as speeds where the tip velocities are greater than or equal to about 68 m/s. Some embodiments of the gear may also utilize teeth that also have second extended portions attached to the second sides of the teeth to divert flow of fluid adjacent to the body of the gear to reduce windage losses that occur when the gear rotates.

  12. Testing new submersible pumps for proper sizing and reduced costs

    Energy Technology Data Exchange (ETDEWEB)

    O' Toole, W.P.; O' Brien, J.B.

    1989-02-01

    This paper describes an ongoing program to improve overall submersible pump performance by Thums Long Beach Co., acting as contractor for the City of Long Beach, operator of the Long Beach Unit. Thums Long Beach Co. currently operates 700 submersible pump installations located on four manmade islands and one landfill pier location. The program began with spot testing of submersible pumps for Thums' use. It has evolved to 100% pump testing and the stipulation that only pumps with newly manufactured parts are acceptable. The primary goals of this program are to increase well production and to lower lifting costs. Critical to these goals is increasing the average length of run by using accurate pump-performance data to design equipment and by rejecting defective pumps before they are run. Increased production is realized from better designs. Lower lifting costs result from using more efficient pumps and a reduced frequency of pulling submersible equipment.

  13. Testing new submersible pumps for proper sizing and reduced costs

    Energy Technology Data Exchange (ETDEWEB)

    O' Toole, W.P.; O' Brien, J.B.

    1986-01-01

    This paper describes an ongoing program to improve overall submersible pump performance by Thums Long Beach Company, acting as Contractor of the City of Long Beach, Operator of the Long Beach Unit. Thums Long Beach Company currently operates 700 submersible pump installations located on four man-made islands and one land fill pier location. The program began with spot testing of submersible pumps for Thums' use. It has evolved to 100 percent pump testing and the stipulation that only pumps with newly manufactured parts are acceptable. The primary goals of this program are to increase well production and lower lifting costs. Critical to these goals is increasing the average length of run by using accurate pump performance data to design equipment and by rejecting defective pumps before they are run. Increased production is realized from better designs. Lower lifting costs result from utilizing higher efficiency pumps and a reduced frequency of pulling submersible equipment.

  14. Insights into the self-reproduction of oleate vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Stano, P [' Enrico Fermi' Centre, Compendio Viminale, 00184 Rome (Italy); Wehrli, E [Electron Microscopy Centre (EMEZ), Applied Physics Institute, ETH Hoenggerberg, 8093 Zurich (Switzerland); Luisi, P L [Biology Department, University of RomaTre, Viale Marconi 446, 00146 Rome (Italy)

    2006-08-23

    In view of the importance of vesicles as models for early cells, several groups have started work looking for conditions under which vesicles can undergo growth and division. Evidence for growth and division has been obtained with the help of ferritin-labelled vesicles; furthermore, it has been shown that in such processes the vesicle size distribution is largely conserved. In both cases, the data suggest that the process under study is mainly characterized by vesicle growth and eventually division into daughter vesicles. However, direct evidence for vesicle division has not been obtained. In this paper, mostly based on freeze-fracture electron microscopy, we describe conditions under which for the first time division intermediates can be trapped in the form of twin vesicles. This finding, together with supporting dynamic light scattering and fluorescence investigations, permits us to establish some additional points in the mechanism of vesicle self-reproduction.

  15. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  16. Sizing Optimization and Strength Analysis for Spread-type Gear Reducers

    Directory of Open Access Journals (Sweden)

    Wei-Hsuan Hsu

    2014-08-01

    Full Text Available A reducer is now developed towards the trend of customization service and cost-saving. In this study, a sizing program for the reducer has been developed in order to replace the manual sizing process. We aim at the total center distance of the gear reducer for optimization to reduce gear volume and weight. Also, we checked constrains such as, tooth root bending, tooth contact strength, gear shaft endangered cross-section, bearing life, gear shaft deflection, and torsion angle deformation, etc., to obtain reliable drive strength. Comparisons of sizes and weights before and after optimization confirm that the purpose for reducing production cost is achieved.

  17. Preparation and Evaluation of Nano-vesicles of Brimonidine Tartrate as an Ocular Drug Delivery System.

    Science.gov (United States)

    Prabhu, P; Nitish, Kumar R; Koland, M; Harish, Nm; Vijayanarayan, K; Dhondge, G; Charyulu, Rn

    2010-10-01

    The objective of the present investigation was to design a vesicular formulation of brimonidine tartrate and evaluate its ability to reduce the dosing frequency and improve the therapeutic efficacy of the drug. Nano-vesicles of brimonidine tartrate were prepared by film hydration method. The prepared vesicles were evaluated for photomicroscopic characteristics, entrapment efficiency, in vitro, and ex-in vitro drug release and in vivo intraocular pressure (IOP) lowering activity. The methods employed for preparation of vesicles produced nano vesicles of acceptable shape and size. The in vitro, and ex-in vitro drug release studies showed that there was slow and prolonged release of the drug, which followed zero-order kinetics. The IOP-lowering activity of nano vesicles was determined and compared with that of pure drug solution and showed that the IOP-lowering action of nano-vesicles sustained for a longer period of time. Stability studies revealed that the vesicle formulations were stable at the temperature range of 2-8°C, with no change in shape and drug content. The results of the study indicate that it is possible to develop a safe and physiologically effective topical formulation that is also convenient for patients.

  18. Extracellular vesicles in cardiovascular homeostasis and disease.

    Science.gov (United States)

    Hutcheson, Joshua D; Aikawa, Elena

    2018-02-19

    Extracellular vesicles have emerged as one of the most important means through which cells interact with each other and the extracellular environment, but extracellular vesicle research remains challenging due to their small size, limited amount of material required for traditional molecular biology assays and inconsistency in the methods of their isolation. The advent of new technologies and standards in the field, however, have led to increased mechanistic insight into extracellular vesicle function. Herein, the latest studies on the role of extracellular vesicles in cardiovascular physiology and disease are discussed. Extracellular vesicles help control cardiovascular homeostasis and remodelling by mediating communication between cells and directing alterations in the extracellular matrix to respond to changes in the environment. The message carried from the parent cell to extracellular space can be intended for both local (within the same tissue) and distal (downstream of blood flow) targets. Pathological cargo loaded within extracellular vesicles could further result in various diseases. On the contrary, new studies indicate that injection of extracellular vesicles obtained from cultured cells into diseased tissues can promote restoration of normal tissue function. Extracellular vesicles are an integral part of cell and tissue function, and harnessing the properties inherent to extracellular vesicles may provide a therapeutic strategy to promote tissue regeneration.

  19. Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

    Energy Technology Data Exchange (ETDEWEB)

    Wasik, Anita A.; Dumont, Vincent [Department of Pathology, University of Helsinki, 00014 Helsinki (Finland); Tienari, Jukka [Department of Pathology, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, 05850 Hyvinkää (Finland); Nyman, Tuula A. [Institute of Biotechnology, University of Helsinki, 00014 Helsinki (Finland); Fogarty, Christopher L.; Forsblom, Carol; Lehto, Markku [Folkhälsan Institute of Genetics, Folkhälsan Research Center, 00290 Helsinki (Finland); Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki (Finland); Diabetes& Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki (Finland); Lehtonen, Eero [Department of Pathology, University of Helsinki, 00014 Helsinki (Finland); Laboratory Animal Centre, University of Helsinki, 00014 Helsinki (Finland); Groop, Per-Henrik [Folkhälsan Institute of Genetics, Folkhälsan Research Center, 00290 Helsinki (Finland); Abdominal Center Nephrology, University of Helsinki and Helsinki University Hospital, 000290 Helsinki (Finland); Diabetes& Obesity Research Program, Research Program´s Unit, 00014 University of Helsinki (Finland); Baker IDI Heart & Diabetes Institute, 3004 Melbourne (Australia); Lehtonen, Sanna, E-mail: sanna.h.lehtonen@helsinki.fi [Department of Pathology, University of Helsinki, 00014 Helsinki (Finland)

    2017-01-15

    Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.

  20. Dietary Nitrate Supplementation Reduces Circulating Platelet-Derived Extracellular Vesicles in Coronary Artery Disease Patients on Clopidogrel Therapy: A Randomised, Double-Blind, Placebo-Controlled Study.

    Science.gov (United States)

    Burnley-Hall, Nicholas; Abdul, Fairoz; Androshchuk, Vitaliy; Morris, Keith; Ossei-Gerning, Nick; Anderson, Richard; Rees, D Aled; James, Philip E

    2018-01-01

    Extracellular vesicles (EVs) are implicated in the pathogenesis of cardiovascular disease (CVD). Specifically, platelet-derived EVs are highly pro-coagulant, promoting thrombin generation and fibrin clot formation. Nitrate supplementation exerts beneficial effects in CVD, via an increase in nitric oxide (NO) bioavailability. Clopidogrel is capable of producing NO-donating compounds, such as S-nitrosothiols (RSNO) in the presence of nitrite and low pH. The aim of this study was to assess the effect of nitrate supplementation with versus without clopidogrel therapy on circulating EVs in coronary artery disease (CAD) patients. In this randomized, double-blind, placebo-controlled study, CAD patients with (n = 10) or without (n = 10) clopidogrel therapy received a dietary nitrate supplement (SiS nitrate gel) or identical placebo. NO metabolites and platelet activation were measured using ozone-based chemiluminescence and multiple electrode aggregometry. EV concentration and origin were determined using nanoparticle tracking analysis and time-resolved fluorescence. Following nitrate supplementation, plasma RSNO was elevated (4.7 ± 0.8 vs 0.2 ± 0.5 nM) and thrombin-receptor mediated platelet aggregation was reduced (-19.9 ± 6.0 vs 4.0 ± 6.4 U) only in the clopidogrel group compared with placebo. Circulating EVs were significantly reduced in this group (-1.183e11 ± 3.15e10 vs -9.93e9 ± 1.84e10 EVs/mL), specifically the proportion of CD41+ EVs (-2,120 ± 728 vs 235 ± 436 RFU [relative fluorescence unit]) compared with placebo. In vitro experiments demonstrated clopidogrel-SNO can reduce platelet-EV directly (6.209e10 ± 4.074e9 vs 3.94e11 ±  1.91e10 EVs/mL). In conclusion, nitrate supplementation reduces platelet-derived EVs in CAD patients on clopidogrel therapy, increasing patient responsiveness to clopidogrel. Nitrate supplementation may represent a novel approach to moderating the risk of thrombus formation in

  1. Alternative methods for characterization of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Fatemeh eMomen-Heravi

    2012-09-01

    Full Text Available Extracellular vesicles are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize Extracellular vesicles. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some Extracellular vesicles -specific evidence. Characterization of Extracellular vesicles has also recently seen many advances with the use of Nanoparticle Tracking Analysis (NTA, flow cytometry, cryo-EM instruments and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.

  2. Apoptotic Bodies: Selective Detection in Extracellular Vesicles.

    Science.gov (United States)

    Hauser, Paul; Wang, Sha; Didenko, Vladimir V

    2017-01-01

    Normal and dying cells release various types of membrane-bound vesicles including microvesicles, exosomes, and apoptotic bodies. These vesicles play important roles in intercellular communication and signal transduction. However, their diverse forms and subtypes fluctuate in size and other properties. In result current purification approaches do not fully discriminate between different categories of extracellular vesicles. Here, we present a fluorescence technique that specifically identifies apoptotic bodies in preparations of microvesicles, exosomes, and other extracellular vesicles.The approach exclusively labels the vesicles that contain DNA with 5'PO 4 blunt-ended DNA breaks, such as those produced by the apoptotic CAD nuclease during apoptotic DNA degradation. The technique can be useful in studies of apoptosis involving microvesicles and exosomes.

  3. DSCR1/RCAN1 regulates vesicle exocytosis and fusion pore kinetics: implications for Down syndrome and Alzheimer's disease.

    Science.gov (United States)

    Keating, Damien J; Dubach, Daphne; Zanin, Mark P; Yu, Yong; Martin, Katherine; Zhao, Yu-Feng; Chen, Chen; Porta, Sílvia; Arbonés, Maria L; Mittaz, Laureane; Pritchard, Melanie A

    2008-04-01

    Genes located on chromosome 21, over-expressed in Down syndrome (DS) and Alzheimer's disease (AD) and which regulate vesicle trafficking, are strong candidates for involvement in AD neuropathology. Regulator of calcineurin activity 1 (RCAN1) is one such gene. We have generated mutant mice in which RCAN1 is either over-expressed (RCAN1(ox)) or ablated (Rcan1-/-) and examined whether exocytosis from chromaffin cells, a classic cellular model of neuronal exocytosis, is altered using carbon fibre amperometry. We find that Rcan1 regulates the number of vesicles undergoing exocytosis and the speed at which the vesicle fusion pore opens and closes. Cells from both Rcan1-/- and RCAN1(ox) mice display reduced levels of exocytosis. Changes in single-vesicle fusion kinetics are also evident resulting in the less catecholamine released per vesicle with increasing Rcan1 expression. Acute calcineurin inhibition did not replicate the effect of RCAN1 overexpression. These changes are not due to alterations in Ca2+ entry or the readily releasable vesicle pool size. Thus, we illustrate a novel regulator of vesicle exocytosis, Rcan1, which influences both exocytotic rate and vesicle fusion kinetics. If Rcan1 functions similarly in neurons then overexpression of this protein, as occurs in DS and AD brains, will reduce both the number of synaptic vesicles undergoing exocytosis and the amount of neurotransmitter released per fusion event. This has direct implications for the pathogenesis of these diseases as sufficient levels of neurotransmission are required for synaptic maintenance and the prevention of neurodegeneration and vesicle trafficking defects are the earliest hallmark of AD neuropathology.

  4. Small portion sizes in worksite cafeterias: do they help consumers to reduce their food intake?

    NARCIS (Netherlands)

    Vermeer, W.M.; Steenhuis, I.H.M.; Leeuwis, F.H.; Heijmans, M.W.; Seidell, J.C.

    2011-01-01

    Background:Environmental interventions directed at portion size might help consumers to reduce their food intake.Objective:To assess whether offering a smaller hot meal, in addition to the existing size, stimulates people to replace their large meal with a smaller meal.Design:Longitudinal randomized

  5. A simulated Experiment for Sampling Soil Micriarthropods to Reduce Sample Size

    OpenAIRE

    Tamura, Hiroshi

    1987-01-01

    An experiment was conducted to examine a possibility of reducing the necessary sample size in a quantitative survey on soil microarthropods, using soybeans instead of animals. An artificially provided, intensely aggregated distribution pattern of soybeans was easily transformed to the random pattern by stirring the substrate, which is soil in a large cardboard box. This enabled the necessary sample size to be greatly reduced without sacrificing the statistical reliability. A new practical met...

  6. Ready-made chromatography columns for extracellular vesicle isolation from plasma

    Directory of Open Access Journals (Sweden)

    Joanne Louise Welton

    2015-03-01

    Full Text Available Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc. that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio. The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.

  7. Lipid Vesicle Shape Analysis from Populations Using Light Video Microscopy and Computer Vision

    OpenAIRE

    Jernej Zupanc; Barbara Drašler; Sabina Boljte; Veronika Kralj-Iglič; Aleš Iglič; Deniz Erdogmus; Damjana Drobne

    2014-01-01

    We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1-50 µm in diameter). For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their ...

  8. Reduced body size and cub recruitment in polar bears associated with sea ice decline

    Science.gov (United States)

    Rode, K.D.; Amstrup, Steven C.; Regehr, E.V.

    2010-01-01

    Rates of reproduction and survival are dependent upon adequate body size and condition of individuals. Declines in size and condition have provided early indicators of population decline in polar bears (Ursus maritimus) near the southern extreme of their range. We tested whether patterns in body size, condition, and cub recruitment of polar bears in the southern Beaufort Sea of Alaska were related to the availability of preferred sea ice habitats and whether these measures and habitat availability exhibited trends over time, between 1982 and 2006. The mean skull size and body length of all polar bears over three years of age declined over time, corresponding with long-term declines in the spatial and temporal availability of sea ice habitat. Body size of young, growing bears declined over time and was smaller after years when sea ice availability was reduced. Reduced litter mass and numbers of yearlings per female following years with lower availability of optimal sea ice habitat, suggest reduced reproductive output and juvenile survival. These results, based on analysis of a longterm data set, suggest that declining sea ice is associated with nutritional limitations that reduced body size and reproduction in this population. ?? 2010 by the Ecological Society of America.

  9. Reduced body size and cub recruitment in polar bears associated with sea ice decline.

    Science.gov (United States)

    Rode, Karyn D; Amstrup, Steven C; Regehr, Eric V

    2010-04-01

    Rates of reproduction and survival are dependent upon adequate body size and condition of individuals. Declines in size and condition have provided early indicators of population decline in polar bears (Ursus maritimus) near the southern extreme of their range. We tested whether patterns in body size, condition, and cub recruitment of polar bears in the southern Beaufort Sea of Alaska were related to the availability of preferred sea ice habitats and whether these measures and habitat availability exhibited trends over time, between 1982 and 2006. The mean skull size and body length of all polar bears over three years of age declined over time, corresponding with long-term declines in the spatial and temporal availability of sea ice habitat. Body size of young, growing bears declined over time and was smaller after years when sea ice availability was reduced. Reduced litter mass and numbers of yearlings per female following years with lower availability of optimal sea ice habitat, suggest reduced reproductive output and juvenile survival. These results, based on analysis of a long-term data set, suggest that declining sea ice is associated with nutritional limitations that reduced body size and reproduction in this population.

  10. Differential Responses of Nitrate Reducer Community Size, Structure, and Activity to Tillage Systems▿ †

    Science.gov (United States)

    Chèneby, D.; Brauman, A.; Rabary, B.; Philippot, L.

    2009-01-01

    The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients. PMID:19304827

  11. Chronic early life lead (Pb2+) exposure alters presynaptic vesicle pools in hippocampal synapses.

    Science.gov (United States)

    Guariglia, Sara Rose; Stansfield, Kirstie H; McGlothan, Jennifer; Guilarte, Tomas R

    2016-11-02

    Lead (Pb2+) exposure has been shown to impair presynaptic neurotransmitter release in both in vivo and in vitro model systems. The mechanism by which Pb2+ impairs neurotransmitter release has not been fully elucidated. In previous work, we have shown that Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites in cultured hippocampal neurons. We have also shown that Pb2+ exposure inhibits vesicular release and alters the distribution of presynaptic vesicles in Shaffer Collateral - CA1 synapses of rodents chronically exposed to Pb2+ during development. In the present study, we used transmission electron microscopy to examine presynaptic vesicle pools in Mossy Fiber-CA3 synapses and in Perforant Path-Dentate Gyrus synapses of rats to determine if in vivo Pb2+ exposure altered presynaptic vesicle distribution in these hippocampal regions. Data were analyzed using T-test for each experimental endpoint. We found that Pb2+ exposure significantly reduced the number of vesicles in the readily releasable pool and recycling pool in Mossy Fiber-CA3 terminals. In both Mossy Fiber-CA3 terminals and in Perforant Path-Dentate Gyrus terminals, Pb2+ exposure significantly increased vesicle nearest neighbor distance in all vesicular pools (Rapidly Releasable, Recycling and Resting). We also found a reduction in the size of the postsynaptic densities of CA3 dendrites in the Pb2+ exposed group. In our previous work, we have demonstrated that Pb2+ exposure impairs vesicular release in Shaffer Collateral - CA1 terminals of the hippocampus and that the number of docked vesicles in the presynaptic active zone was reduced. Our current data shows that Pb2+ exposure reduces the number of vesicles that are in proximity to release sites in Mossy Fiber- CA3 terminals. Furthermore, Pb2+ exposure causes presynaptic vesicles to be further from one another, in both Mossy Fiber- CA3 terminals and in Perforant Pathway - Dentate Gyrus terminals, which may interfere with

  12. Reducing size-dispersion in one-pot aqueous synthesis of maghemite nanoparticles.

    Science.gov (United States)

    Drummond, A L; Feitoza, N C; Duarte, G C; Sales, M J A; Silva, L P; Chaker, J A; Bakuzis, A F; Sousa, M H

    2012-10-01

    Nanosized maghemite-like particles with reduced size-distribution were obtained using a one-pot synthesis route in aqueous medium. Forced hydrolysis of iron ions in ammoniac solution led to the formation of magnetite nanoparticles that were oxidized to maghemite in a hydrothermal digestion step that reduced the polydispersity of nanograins. The prepared nanoparticles were characterized by chemical analysis, X-ray diffractometry, magnetization, Raman spectroscopy and transmission electron microscopy measurements. Data showed that 14 nm-sized particles with polydispersity of about 0.14 were produced and, differently from other procedures, neither additional steps nor toxic reagents were needed to reduce size-dispersion or to oxidize magnetite to maghemite. These facts per se turn such nanodevice into a good potential choice for biomedical applications.

  13. Structure of Amphiphilic Terpolymer Raspberry Vesicles

    Directory of Open Access Journals (Sweden)

    Yingying Guo

    2017-07-01

    Full Text Available Terpolymer raspberry vesicles contain domains of different chemical affinities. They are potential candidates as multi-compartment cargo carriers. Their efficacy depends on their stability and load capacity. Using a model star terpolymer system in an aqueous solution, a dissipative particle dynamic (DPD simulation is employed to investigate how equilibrium aggregate structures are affected by polymer concentration and pairwise interaction energy in a solution. It is shown that a critical mass of polymer is necessary for vesicle formation. The free energy of the equilibrium aggregates are calculated and the results show that the transition from micelles to vesicles is governed by the interactions between the longest solvophobic block and the solvent. In addition, the ability of vesicles to encapsulate solvent is assessed. It is found that reducing the interaction energy favours solvent encapsulation, although solvent molecules can permeate through the vesicle’s shell when repulsive interactions among monomers are low. Thus, one can optimize the loading capacity and the release rate of the vesicles by turning pairwise interaction energies of the polymer and the solvent. The ability to predict and control these aspects of the vesicles is an essential step towards designing vesicles for specific purposes.

  14. Soft vesicles in the synthesis of hard materials.

    Science.gov (United States)

    Dong, Renhao; Liu, Weimin; Hao, Jingcheng

    2012-04-17

    Vesicles of surfactants in aqueous solution have received considerable attention because of their use as simple model systems for biological membranes and their applications in various fields including colloids, pharmaceuticals, and materials. Because of their architecture, vesicles could prove useful as "soft" templates for the synthesis of "hard materials". The vesicle phase, however, has been challenging and difficult to work with in the construction of hard materials. In the solution-phase synthesis of various inorganic or macromolecular materials, templating methods provide a powerful strategy to control the size, morphology, and composition of the resulting micro- and nanostructures. In comparison with hard templates, soft templates are generally constructed using amphiphilic molecules, especially surfactants and amphiphilic polymers. These types of compounds offer advantages including the wide variety of available templates, simple fabrication processes under mild conditions, and easy removal of the templates with less damage to the final structures. Researchers have used many ordered molecular aggregates such as vesicles, micelles, liquid crystals, emulsion droplets, and lipid nanotubes as templates or structure-directing agents to control the synthesis or assembly hard micro- and nanomaterials composed from inorganic compounds or polymers. In addition to their range of sizes and morphologies, vesicles present unique structures that can simultaneously supply different microenvironments for the growth and assembly of hard materials: the inner chamber of vesicles, the outer surface of the vesicles, and the space between bilayers. Two main approaches for applying vesicles in the field of hard materials have been explored: (i) in situ synthesis of micro- or nanomaterials within a specific microenvironment by vesicle templating and (ii) the assembly or incorporation of guest materials during the formation of vesicles. This Account provides an in-depth look at

  15. Formation and structural properties of multi-block copolymer vesicles

    Science.gov (United States)

    Wang, Rong; Ma, Shiying

    2014-03-01

    Due to the unique structure, vesicles have attracted considerable attention for their potential applications, such as gene and drug delivery, microcapsules, nanoreactors, cell membrane mimetic, synthetic organelles, etc. By using dissipative particle dynamics, we studied the self-assembly of amphiphilic multi-block copolymer. The phase diagram was constructed by varying the interaction parameters and the composition of the block copolymers. The results show that the vesicles are stable in a large region which is different from the diblock copolymer or triblock copolymer. The structural properties of vesicles can be controlled by varying the interaction parameters and the length of the hydrophobic block. The relationship between the hydrophilic and hydrophobic block length vs the aqueous cavity size and vesicle size are revealed. The copolymers with shorter hydrophobic blocks length or the higher hydrophilicity are more likely to form vesicles with larger aqueous cavity size and vesicle size as well as thinner wall thickness. However, the increase in hydrophobic-block length results to form vesicles with smaller aqueous cavity size and larger vesicle size. Acknowledgments. This work has been supported by NNSFC (No. 21074053) and NBRPC (No. 2010CB923303).

  16. Synaptic Vesicle Endocytosis

    Science.gov (United States)

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

  17. Implantation of cocoa butter reduces egg and hatchling size in Salmo trutta

    NARCIS (Netherlands)

    Hoogenboom, M. O.; Armstrong, J. D.; Miles, M. S.; Burton, T.; Groothuis, T. G. G.; Metcalfe, N. B.

    This study demonstrated that, irrespective of hormone type or dose, administering cocoa butter implants during egg development affected the growth of female brown trout Salmo trutta and reduced the size of their offspring. Cortisol treatment also increased adult mortality. Caution is urged in the

  18. Reduced Sampling Size with Nanopipette for Tapping-Mode Scanning Probe Electrospray Ionization Mass Spectrometry Imaging.

    Science.gov (United States)

    Kohigashi, Tsuyoshi; Otsuka, Yoichi; Shimazu, Ryo; Matsumoto, Takuya; Iwata, Futoshi; Kawasaki, Hideya; Arakawa, Ryuichi

    2016-01-01

    Mass spectrometry imaging (MSI) with ambient sampling and ionization can rapidly and easily capture the distribution of chemical components in a solid sample. Because the spatial resolution of MSI is limited by the size of the sampling area, reducing sampling size is an important goal for high resolution MSI. Here, we report the first use of a nanopipette for sampling and ionization by tapping-mode scanning probe electrospray ionization (t-SPESI). The spot size of the sampling area of a dye molecular film on a glass substrate was decreased to 6 μm on average by using a nanopipette. On the other hand, ionization efficiency increased with decreasing solvent flow rate. Our results indicate the compatibility between a reduced sampling area and the ionization efficiency using a nanopipette. MSI of micropatterns of ink on a glass and a polymer substrate were also demonstrated.

  19. FIJI Macro 3D ART VeSElecT: 3D Automated Reconstruction Tool for Vesicle Structures of Electron Tomograms.

    Directory of Open Access Journals (Sweden)

    Kristin Verena Kaltdorf

    2017-01-01

    Full Text Available Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i in embryonic Danio rerio 4 and 8 days past fertilization (dpf and (ii to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ wild-type and its septin mutant (unc-59(e261. We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261 on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement. This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter. Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles and specificity (true vesicles as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual

  20. Reduced particle size wheat bran is butyrogenic and lowers Salmonella colonization, when added to poultry feed.

    Science.gov (United States)

    Vermeulen, K; Verspreet, J; Courtin, C M; Haesebrouck, F; Ducatelle, R; Van Immerseel, F

    2017-01-01

    Feed additives, including prebiotics, are commonly used alternatives to antimicrobial growth promoters to improve gut health and performance in broilers. Wheat bran is a highly concentrated source of (in)soluble fiber which is partly degraded by the gut microbiota. The aim of the present study was to investigate the potential of wheat bran as such to reduce colonization of the cecum and shedding of Salmonella bacteria in vivo. Also, the effect of particle size was evaluated. Bran with an average reduced particle size of 280μm decreased levels of cecal Salmonella colonization and shedding shortly after infection when compared to control groups and groups receiving bran with larger particle sizes. In vitro fermentation experiments revealed that bran with smaller particle size was fermented more efficiently, with a significantly higher production of butyric and propionic acid, compared to the control fermentation and fermentation of a larger fraction. Fermentation products derived from bran with an average particle size of 280μm downregulated the expression of hilA, an important invasion-related gene of Salmonella. This downregulation was reflected in an actual lowered invasive potential when Salmonella bacteria were pretreated with the fermentation products derived from the smaller bran fraction. These data suggest that wheat bran with reduced particle size can be a suitable feed additive to help control Salmonella infections in broilers. The mechanism of action most probably relies on a more efficient fermentation of this bran fraction and the consequent increased production of short chain fatty acids (SCFA). Among these SCFA, butyric and propionic acid are known to reduce the invasion potential of Salmonella bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles.

    Directory of Open Access Journals (Sweden)

    Taulant Bacaj

    2015-10-01

    Full Text Available In forebrain neurons, Ca(2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca(2+-independent, mutations in Ca(2+-binding sequences of synaptotagmin-1 or synaptotagmin-7--which are contained in flexible top-loop sequences of their C2 domains--blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca(2+ triggering of different phases of release.

  2. Three-component vesicle aggregation driven by adhesion interactions between Au nanoparticles and polydopamine-coated nanotubes.

    Science.gov (United States)

    Jin, Haibao; Zhou, Yongfeng; Huang, Wei; Zheng, Yongli; Zhu, Xinyuan; Yan, Deyue

    2014-06-11

    Large-scale and robust vesicle aggregates were obtained through molecular recognition among cell-sized polymer vesicles, carbon nanotubes and AuNPs, driven by adhesion interactions between Au and polydopamine. Vesicle fusion was effectively avoided in this three-component vesicle aggregation process.

  3. Novel joint selection methods can reduce sample size for rheumatoid arthritis clinical trials with ultrasound endpoints.

    Science.gov (United States)

    Allen, John C; Thumboo, Julian; Lye, Weng Kit; Conaghan, Philip G; Chew, Li-Ching; Tan, York Kiat

    2017-10-03

    To determine whether novel methods of selecting joints through (i) ultrasonography (individualized-ultrasound [IUS] method), or (ii) ultrasonography and clinical examination (individualized-composite-ultrasound [ICUS] method) translate into smaller rheumatoid arthritis (RA) clinical trial sample sizes when compared to existing methods utilizing predetermined joint sites for ultrasonography. Cohen's effect size (ES) was estimated (ES^) and a 95% CI (ES^L, ES^U) calculated on a mean change in 3-month total inflammatory score for each method. Corresponding 95% CIs [nL(ES^U), nU(ES^L)] were obtained on a post hoc sample size reflecting the uncertainty in ES^. Sample size calculations were based on a one-sample t-test as the patient numbers needed to provide 80% power at α = 0.05 to reject a null hypothesis H0 : ES = 0 versus alternative hypotheses H1 : ES = ES^, ES = ES^L and ES = ES^U. We aimed to provide point and interval estimates on projected sample sizes for future studies reflecting the uncertainty in our study ES^S. Twenty-four treated RA patients were followed up for 3 months. Utilizing the 12-joint approach and existing methods, the post hoc sample size (95% CI) was 22 (10-245). Corresponding sample sizes using ICUS and IUS were 11 (7-40) and 11 (6-38), respectively. Utilizing a seven-joint approach, the corresponding sample sizes using ICUS and IUS methods were nine (6-24) and 11 (6-35), respectively. Our pilot study suggests that sample size for RA clinical trials with ultrasound endpoints may be reduced using the novel methods, providing justification for larger studies to confirm these observations. © 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

  4. Immunotherapeutic potential of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Bin eZhang

    2014-10-01

    Full Text Available Extracellular vesicles or EVs is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarisation to Tregs, immune suppression and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

  5. Chronic administration of OB protein decreases food intake by selectively reducing meal size in female rats.

    Science.gov (United States)

    Eckel, L A; Langhans, W; Kahler, A; Campfield, L A; Smith, F J; Geary, N

    1998-07-01

    The mechanisms by which OB protein controls food intake and energy balance are unknown. Therefore, we investigated the effects of a novel modified human recombinant OB protein (Mod-OB) on spontaneous feeding patterns, body weight, running wheel activity, and ovarian cycling in female rats. Mod-OB or vehicle was injected (4 mg . kg-1 . day-1 sc) for 2 ovarian cycles (8 days) using a within-subjects design. Observations were continued for five ovarian cycles after injections; treatments were then reversed. Mod-OB reduced food intake approximately 20% from injection day 1 to postinjection day 2. Body weight was reduced from injection day 3 to postinjection day 15 (maximum decrease, 25 +/- 4 g, postinjection days 3 and 4). Food intake was reduced due to decreases in nocturnal meal size, which appeared to be superimposed on the normal pattern of spontaneous feeding (i.e., reductions in meal size at estrus). Mod-OB did not significantly affect diurnal food intake or meal patterns, failed to alter wheel running, and did not disrupt the rats' ovarian cycles. We conclude that chronically administered Mod-OB reduces food intake in female rats by selectively affecting the mechanisms controlling meal size.

  6. Fusion of Nonionic Vesicles

    DEFF Research Database (Denmark)

    Bulut, Sanja; Oskolkova, M. Z.; Schweins, R.

    2010-01-01

    We present an experimental study of vesicle fusion using light and neutron scattering to monitor fusion events. Vesicles are reproducibly formed with an extrusion procedure using an single amphiphile triethylene glycol mono-n-decyl ether in water. They show long-term stability for temperatures ar...... a barrier to fusion changing from 15 k(B)T at T = 26 degrees C to 10k(H) T at T = 35 degrees C. These results are compatible with the theoretical predictions using the stalk model of vesicle fusion....

  7. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    Science.gov (United States)

    Erdbrügger, Uta; Le, Thu H

    2016-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles. Copyright © 2016 by the American Society of Nephrology.

  8. Small portion sizes in worksite cafeterias: do they help consumers to reduce their food intake?

    Science.gov (United States)

    Vermeer, W M; Steenhuis, I H M; Leeuwis, F H; Heymans, M W; Seidell, J C

    2011-09-01

    Environmental interventions directed at portion size might help consumers to reduce their food intake. To assess whether offering a smaller hot meal, in addition to the existing size, stimulates people to replace their large meal with a smaller meal. Longitudinal randomized controlled trial assessing the impact of introducing small portion sizes and pricing strategies on consumer choices. In all, 25 worksite cafeterias and a panel consisting of 308 consumers (mean age=39.18 years, 50% women). A small portion size of hot meals was offered in addition to the existing size. The meals were either proportionally priced (that is, the price per gram was comparable regardless of the size) or value size pricing was employed. Daily sales of small and the total number of meals, consumers' self-reported compensation behavior and frequency of purchasing small meals. The ratio of small meals sales in relation to large meals sales was 10.2%. No effect of proportional pricing was found B=-0.11 (0.33), P=0.74, confidence interval (CI): -0.76 to 0.54). The consumer data indicated that 19.5% of the participants who had selected a small meal often-to-always purchased more products than usual in the worksite cafeteria. Small meal purchases were negatively related to being male (B=-0.85 (0.20), P=0.00, CI: -1.24 to -0.46, n=178). When offering a small meal in addition to the existing size, a percentage of consumers that is considered reasonable were inclined to replace the large meal with the small meal. Proportional prices did not have an additional effect. The possible occurrence of compensation behavior is an issue that merits further attention.

  9. Reduced clot debris size using standing waves formed via high intensity focused ultrasound

    Science.gov (United States)

    Guo, Shifang; Du, Xuan; Wang, Xin; Lu, Shukuan; Shi, Aiwei; Xu, Shanshan; Bouakaz, Ayache; Wan, Mingxi

    2017-09-01

    The feasibility of utilizing high intensity focused ultrasound (HIFU) to induce thrombolysis has been demonstrated previously. However, clinical concerns still remain related to the clot debris produced via fragmentation of the original clot potentially being too large and hence occluding downstream vessels, causing hazardous emboli. This study investigates the use of standing wave fields formed via HIFU to disintegrate the thrombus while achieving a reduced clot debris size in vitro. The results showed that the average diameter of the clot debris calculated by volume percentage was smaller in the standing wave mode than in the travelling wave mode at identical ultrasound thrombolysis settings. Furthermore, the inertial cavitation dose was shown to be lower in the standing wave mode, while the estimated cavitation bubble size distribution was similar in both modes. These results show that a reduction of the clot debris size with standing waves may be attributed to the particle trapping of the acoustic potential well which contributed to particle fragmentation.

  10. Concentration Effect of Reducing Agents on Green Synthesis of Gold Nanoparticles: Size, Morphology, and Growth Mechanism

    Science.gov (United States)

    Kim, Hyun-seok; Seo, Yu Seon; Kim, Kyeounghak; Han, Jeong Woo; Park, Youmie; Cho, Seonho

    2016-04-01

    Under various concentration conditions of reducing agents during the green synthesis of gold nanoparticles (AuNPs), we obtain the various geometry (morphology and size) of AuNPs that play a crucial role in their catalytic properties. Through both theoretical and experimental approaches, we studied the relationship between the concentration of reducing agent (caffeic acid) and the geometry of AuNPs. As the concentration of caffeic acid increases, the sizes of AuNPs were decreased due to the adsorption and stabilizing effect of oxidized caffeic acids (OXCAs). Thus, it turns out that optimal concentration exists for the desired geometry of AuNPs. Furthermore, we investigated the growth mechanism for the green synthesis of AuNPs. As the caffeic acid is added and adsorbed on the surface of AuNPs, the aggregation mechanism and surface free energy are changed and consequently resulted in the AuNPs of various geometry.

  11. Gram-negative and Gram-positive bacterial extracellular vesicles.

    Science.gov (United States)

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Complement factor 5 blockade reduces porcine myocardial infarction size and improves immediate cardiac function.

    Science.gov (United States)

    Pischke, Soeren E; Gustavsen, A; Orrem, H L; Egge, K H; Courivaud, F; Fontenelle, H; Despont, A; Bongoni, A K; Rieben, R; Tønnessen, T I; Nunn, M A; Scott, H; Skulstad, H; Barratt-Due, A; Mollnes, T E

    2017-05-01

    Inhibition of complement factor 5 (C5) reduced myocardial infarction in animal studies, while no benefit was found in clinical studies. Due to lack of cross-reactivity of clinically used C5 antibodies, different inhibitors were used in animal and clinical studies. Coversin (Ornithodoros moubata complement inhibitor, OmCI) blocks C5 cleavage and binds leukotriene B4 in humans and pigs. We hypothesized that inhibition of C5 before reperfusion will decrease infarct size and improve ventricular function in a porcine model of myocardial infarction. In pigs (Sus scrofa), the left anterior descending coronary artery was occluded (40 min) and reperfused (240 min). Coversin or placebo was infused 20 min after occlusion and throughout reperfusion in 16 blindly randomized pigs. Coversin significantly reduced myocardial infarction in the area at risk by 39% (p = 0.03, triphenyl tetrazolium chloride staining) and by 19% (p = 0.02) using magnetic resonance imaging. The methods correlated significantly (R = 0.92, p infarcted area at risk by coversin treatment. Coversin ablated plasma C5 activation throughout the reperfusion period and decreased myocardial C5b-9 deposition, while neither plasma nor myocardial LTB4 were significantly reduced. Coversin substantially reduced the size of infarction, improved ventricular function, and attenuated interleukin-1β and E-selectin in this porcine model by inhibiting C5. We conclude that inhibition of C5 in myocardial infarction should be reconsidered.

  13. Morphine Reduces Myocardial Infarct Size via Heat Shock Protein 90 in Rodents

    Directory of Open Access Journals (Sweden)

    Bryce A. Small

    2015-01-01

    Full Text Available Opioids reduce injury from myocardial ischemia-reperfusion in humans. In experimental models, this mechanism involves GSK3β inhibition. HSP90 regulates mitochondrial protein import, with GSK3β inhibition increasing HSP90 mitochondrial content. Therefore, we determined whether morphine-induced cardioprotection is mediated by HSP90 and if the protective effect is downstream of GSK3β inhibition. Male Sprague-Dawley rats, aged 8–10 weeks, were subjected to an in vivo myocardial ischemia-reperfusion injury protocol involving 30 minutes of ischemia followed by 2 hours of reperfusion. Hemodynamics were continually monitored and myocardial infarct size determined. Rats received morphine (0.3 mg/kg, the GSK3β inhibitor, SB216763 (0.6 mg/kg, or saline, 10 minutes prior to ischemia. Some rats received selective HSP90 inhibitors, radicicol (0.3 mg/kg, or deoxyspergualin (DSG, 0.6 mg/kg alone or 5 minutes prior to morphine or SB216763. Morphine reduced myocardial infarct size when compared to control (42 ± 2% versus 60 ± 1%. This protection was abolished by prior treatment of radicicol or DSG (59 ± 1%, 56 ± 2%. GSK3β inhibition also reduced myocardial infarct size (41 ± 2% with HSP90 inhibition by radicicol or DSG partially inhibiting SB216763-induced infarct size reduction (54 ± 3%, 47 ± 1%, resp.. These data suggest that opioid-induced cardioprotection is mediated by HSP90. Part of this protection afforded by HSP90 is downstream of GSK3β, potentially via the HSP-TOM mitochondrial import pathway.

  14. Sugar-based gemini surfactant with a vesicle-to-micelle transition at acidic pH and a reversible vesicle flocculation near neutral pH

    NARCIS (Netherlands)

    Johnsson, M; Wagenaar, A; Engberts, JBFN

    2003-01-01

    A sugar-based (reduced glucose) gemini surfactant forms vesicles in dilute aqueous solution near neutral pH. At lower pH, there is a vesicle-to-micelle transition within a narrow pH region (pH 6.0-5.6). The vesicles are transformed into large cylindrical micelles that in turn are transformed into

  15. Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins.

    Science.gov (United States)

    Jang, Yeongseon; Choi, Won Tae; Heller, William T; Ke, Zunlong; Wright, Elizabeth R; Champion, Julie A

    2017-09-01

    Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A case of liver hemangioma with markedly reduced tumor size after metformin treatment: a case report.

    Science.gov (United States)

    Ono, Minoru; Sawada, Koji; Okumura, Toshikatsu

    2017-02-01

    A 52-year-old man with a 9-year history of hepatic hemangioma was treated with the anti-diabetic drug metformin, resulting in complete remission of the tumor. In 2006, a hemangioma with diameter of 20 × 25 mm was detected incidentally in the liver. The results of imaging studies including ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) were all compatible with that of hepatic hemangioma. The patient consequently underwent imaging annually from 2006 to 2015. The tumor size increased slightly, to 30 × 35 mm in 2012; however, the general tumor characteristics in imaging were not changed. Beginning May 2012, metformin (750 mg/day) was administered because of an increase in blood sugar and hemoglobin A1c levels. After the start of metformin treatment, the tumor size on US gradually decreased. Finally, in October 2015, the tumor was no longer detected. Dynamic CT study also demonstrated markedly reduced tumor size, with a decrease of 2-3 mm in diameter. These results indicate that metformin treatment strongly suppressed cell proliferation in liver hemangioma. The anti-angiogenic effect of metformin was indicated as a possible cause of the reduction in tumor size.

  17. The Role of Social Norms in the Portion Size Effect: Reducing Normative Relevance Reduces the Effect of Portion Size on Consumption Decisions

    Science.gov (United States)

    Versluis, Iris; Papies, Esther K.

    2016-01-01

    People typically eat more from large portions of food than from small portions. An explanation that has often been given for this so-called portion size effect is that the portion size acts as a social norm and as such communicates how much is appropriate to eat. In this paper, we tested this explanation by examining whether manipulating the relevance of the portion size as a social norm changes the portion size effect, as assessed by prospective consumption decisions. We conducted one pilot experiment and one full experiment in which participants respectively indicated how much they would eat or serve themselves from a given amount of different foods. In the pilot (N = 63), we manipulated normative relevance by allegedly basing the portion size on the behavior of either students of the own university (in-group) or of another university (out-group). In the main experiment (N = 321), we told participants that either a minority or majority of people similar to them approved of the portion size. Results show that in both experiments, participants expected to serve themselves and to eat more from larger than from smaller portions. As expected, however, the portion size effect was less pronounced when the reference portions were allegedly based on the behavior of an out-group (pilot) or approved only by a minority (main experiment). These findings suggest that the portion size indeed provides normative information, because participants were less influenced by it if it communicated the behaviors or values of a less relevant social group. In addition, in the main experiment, the relation between portion size and the expected amount served was partially mediated by the amount that was considered appropriate, suggesting that concerns about eating an appropriate amount indeed play a role in the portion size effect. However, since the portion size effect was weakened but not eliminated by the normative relevance manipulations and since mediation was only partial, other

  18. Phenomena of insulin peak fronting in size exclusion chromatography and strategies to reduce fronting.

    Science.gov (United States)

    Yu, Chi-Ming; Mun, Sungyong; Wang, Nien-Hwa Linda

    2008-05-23

    Insulin peak fronting in size exclusion chromatography (SEC) results in more than 10% yield loss in the production of insulin. The goal of this study is to understand the mechanisms of peak fronting and to develop strategies to reduce fronting and increase insulin yield. Chromatography experiments ruled out pressure surge, viscous fingering, and adsorption as the cause for peak fronting. Theoretical analysis based on a general rate model indicated that reversible dimerization is the major cause for peak fronting and reducing the dimerization equilibrium constant is the most effective method for reducing fronting. Two strategies were developed and tested to reduce the degree of dimer formation. The first strategy was to use 0.1N acetic acid as the presaturant and eluent. The second strategy was to use 0.8 or 2.8N acetic acid in 20vol.% denatured ethanol as the mobile phase. The experimental results showed that both strategies can reduce insulin peak fronting in SEC, maintain desired product purity, and increase insulin yield to higher than 98%.

  19. Can reduced size of metals induce hydrogen absorption: ZrAl{sub 2} case

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, I., E-mail: izi@bgu.ac.il [Department of Nuclear Engineering, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel); Deledda, S. [Physics Department, Institute for Energy Technology, P.O. Box 40, NO-2027 Kjeller (Norway); Bereznitsky, M. [Department of Nuclear Engineering, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel); Yeheskel, O. [Nuclear Research Center - Negev, P.O. Box 9001, Beer Sheva 84190 (Israel); Filipek, S.M. [Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw (Poland); Mogilyanski, D.; Kimmel, G. [Institute for Applied Research, P.O. Box 653, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel); Hauback, B.C. [Physics Department, Institute for Energy Technology, P.O. Box 40, NO-2027 Kjeller (Norway)

    2011-09-15

    Research highlights: > 15 nm particles of ZrAl{sub 2} and Zr(Al{sub 0.5}Co{sub 0.5}){sub 2} are obtained by attrition and cryomilling. > ZrAl{sub 2} nanoparticles remain inert to hydrogen absorption up to pressure of {approx}2 GPa. > Zr(Al{sub 0.5}Co{sub 0.5}){sub 2} nanoparticles exhibit reduced hydrogen absorption as compared to the corresponding bulk compounds. - Abstract: The hydrogen absorption ability of the non-absorbing Al-rich ZrAl{sub 2} compound was examined after reducing its particles-size to the nanometer regime. The hydrogen abstinence of bulk ZrAl{sub 2} has been previously related to its excessive elastic shear stiffening. The particle size of ZrAl{sub 2} was reduced by attrition milling and cryomilling. The minimal average particle size was estimated from powder X-ray diffraction analysis to be in the range of 10-20 nm. The hydrogen absorption of the milled compounds was measured in different hydrogenation systems at hydrogen pressures between {approx}6 MPa and {approx}2 GPa. In all the cases the hydrogen absorption was negligible. In addition, there was a reduction of the hydrogen absorption capacity of nanosized Zr(Al{sub 0.5}Co{sub 0.5}){sub 2} as compared to the corresponding bulk compound at the same conditions. We suggest, in view of our and other results, that no significant improvement of the thermodynamics (unlike the kinetics) of the hydrogen absorption can be achieved via the nanoparticle avenue.

  20. Grain dissection as a grain size reducing mechanism during ice microdynamics

    Science.gov (United States)

    Steinbach, Florian; Kuiper, Ernst N.; Eichler, Jan; Bons, Paul D.; Drury, Martin R.; Griera, Albert; Pennock, Gill M.; Weikusat, Ilka

    2017-04-01

    Ice sheets are valuable paleo-climate archives, but can lose their integrity by ice flow. An understanding of the microdynamic mechanisms controlling the flow of ice is essential when assessing climatic and environmental developments related to ice sheets and glaciers. For instance, the development of a consistent mechanistic grain size law would support larger scale ice flow models. Recent research made significant progress in numerically modelling deformation and recrystallisation mechanisms in the polycrystalline ice and ice-air aggregate (Llorens et al., 2016a,b; Steinbach et al., 2016). The numerical setup assumed grain size reduction is achieved by the progressive transformation of subgrain boundaries into new high angle grain boundaries splitting an existing grain. This mechanism is usually termed polygonisation. Analogue experiments suggested, that strain induced grain boundary migration can cause bulges to migrate through the whole of a grain separating one region of the grain from another (Jessell, 1986; Urai, 1987). This mechanism of grain dissection could provide an alternative grain size reducing mechanism, but has not yet been observed during ice microdynamics. In this contribution, we present results using an updated numerical approach allowing for grain dissection. The approach is based on coupling the full field theory crystal visco-plasticity code (VPFFT) of Lebensohn (2001) to the multi-process modelling platform Elle (Bons et al., 2008). VPFFT predicts the mechanical fields resulting from short strain increments, dynamic recrystallisation process are implemented in Elle. The novel approach includes improvements to allow for grain dissection, which was topologically impossible during earlier simulations. The simulations are supported by microstructural observations from NEEM (North Greenland Eemian Ice Drilling) ice core. Mappings of c-axis orientations using the automatic fabric analyser and full crystallographic orientations using electron

  1. Reducing Data Size Inequality during Finite Element Model Separation into Superelements

    Directory of Open Access Journals (Sweden)

    Yu. V. Berchun

    2015-01-01

    Full Text Available The work considers two methods of automatic separation of final element model into super-elements to decrease computing resource demand when solving the linearly - elastic problems of solid mechanics. The first method represents an algorithm to separate a final element grid into simply connected sub-regions according to the set specific number of nodes in the super-element. The second method is based on the generation of a super-element with the set specific data size of the coefficient matrix of the system of equations of the internal nodes balance, which are eliminated during super-element transformation. Both methods are based on the theory of graphs. The data size of a matrix of coefficients is assessed on the assumption that the further solution of a task will use Holetsky’s method. Before assessment of data size, a KatkhillaMackey's (Cuthill-McKee algorithm renumbers the internal nodes of a super-element both to decrease a profile width of the appropriate matrix of the system of equations of balance and to reduce the number of nonzero elements. Test examples show work results of abovementioned methods compared in terms of inequality of generated super-element separation according to the number of nodes and data size of the coefficient matrix of the system of equations of the internal nodes balance. It is shown that the offered approach provides smaller inequality of data size of super-element matrixes, with slightly increasing inequality by the number of tops.

  2. Is there a critical endometrioma size associated with reduced ovarian responsiveness in assisted reproduction techniques?

    Science.gov (United States)

    Coccia, Maria Elisabetta; Rizzello, Francesca; Barone, Stefano; Pinelli, Sara; Rapalini, Erika; Parri, Cristiana; Caracciolo, Domenico; Papageorgiou, Savvas; Cima, Gianpaolo; Gandini, Loredana

    2014-08-01

    This study investigated the relationships between ovarian endometrioma size, ovarian responsiveness and the number of retrieved oocytes following ovarian stimulation. A prospective study was conducted in a public clinical assisted reproduction centre. A total of 64 infertile women with monolateral endometriomas undergoing IVF or intracytoplasmic sperm injection were included in the study. The total number of follicles, number of follicles ≥ 16 mm and number of oocytes retrieved of ovaries containing endometrioma and normal ovaries were compared. Multivariate linear regression was used to assess whether number of follicles and collected oocytes varied by endometrioma size, age, basal FSH concentration. Significantly lower numbers of follicles ≥ 16 mm (P = 0.024) and oocytes retrieved (P = 0.001) in the ovaries containing endometrioma were observed. In patients with endometriomas ≥ 30 mm, endometrioma size was the most influential contributor to the total number of follicles and oocytes retrieved. Ovarian endometriomas result in reduced response to ovarian stimulation, compared with the response of the contralateral normal ovary in the same individual. In case of endometriomas Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  3. Chronic administration of OB protein decreases food intake by selectively reducing meal size in male rats.

    Science.gov (United States)

    Kahler, A; Geary, N; Eckel, L A; Campfield, L A; Smith, F J; Langhans, W

    1998-07-01

    The potent hypophagic effect of OB protein (OB) is well established, but the mechanism of this effect is largely unknown. We investigated the effects of chronic administration of a novel modified recombinant human OB (Mod-OB) with a prolonged half-life (>48 h) on ad libitum food intake, spontaneous meal patterns, and body weight in 24 adult, male Sprague-Dawley rats (body weight at study onset: 292 g). Single daily subcutaneous injections of Mod-OB (4 mg/kg daily) for 8 consecutive days significantly reduced ad libitum food intake compared with vehicle injections from injection day 3 through postinjection day 3. Mod-OB-injected rats ate between 4.5 and 7.1 g (or 13-20%) per day less than controls, with the reduction primarily occurring during the dark period. Body weight gain was significantly decreased in response to Mod-OB from injection day 8 until postinjection day 4, with a maximum difference of 24 g on postinjection day 3. The reduction of food intake by Mod-OB was mainly due to a 21-34% decrease in nocturnal spontaneous meal size. There was no significant effect of Mod-OB on nocturnal meal frequency or duration. Mod-OB also did not reliably affect the size, duration, or frequency of diurnal meals. Mod-OB-injected rats displayed no compensatory hyperphagia after the injection period. These results indicate that chronically administered OB selectively affects the mechanisms controlling meal size in male rats.

  4. ACECLOFENAC ENCAPSULATED ETHANOLIC NANO-VESICLES FOR EFFECTIVE TREATMENT OF OSTEOART HRITIS

    OpenAIRE

    Arvinder Kaur et al

    2012-01-01

    In the present study, ethanolic nanovesicles of Aceclofenac developed for the site specific delivery to joints for effective treatment of osteoarthritis. Ethanolic nano-vesicles were prepared by solvent dispersion method. Vesicles were characterized for vesicular size, surface morphology, size and size distribution, zeta potential, entrapment efficiency. Formulations were also evaluated for drug-vesicle (excipients) interaction, in vitro permeation, in vitro deposition. The TEM showed dark ve...

  5. Performance of Disk Mill Type Mechanical Grinder for Size Reducing Process of Robusta Roasted Beans

    Directory of Open Access Journals (Sweden)

    Sri Mulato

    2006-12-01

    Full Text Available One of improtant steps in secondary coffee processing that influence on final product quality such as consistency and uniformity is milling process. Usually, Indonesian smallholder used "lumpang" for milling coffee roasted beans to coffee powder product which caused the final product not uniformed and consistent, and low productivity. Milling process of coffee roasted beans can be done by disk mill type mechanical grinder which is used by smallholder for milling several cereals. Indonesian Coffee and Cocoa Research Institute have developed disk mill type grinding machine for milling coffee roasted beans. Objective of this research is to find performance of disk mill type grinding machine for size reducing process of Robusta roasted beans from several size dried beans and roasting level treatments. Robusta dried beans which are taken from dry processing method have 13—14% moisture content (wet basis, 680—685 kg/m3 density, and classified in 3 sizes level. The result showed that the disk mill type of grinding machine could be used for milling Robusta roasted beans. Machine hascapacity 31—54 kg/h on 5,310—5,610 rpm axle rotation and depend on roasting level. Other technical parameters were 91—98% process efficientcy, 19—31 ml/ kg fuel consumption, 0.3—1% slips, 50—55% particles had diameter less than 230 mesh and 38—44% particles had diameter bigger than 100 mesh, 32—38% lightness was increased, 0.6—12.6% density was decreased, and solubility of coffee powder between 28—30%. Cost milling process per kilogram of Robusta roasted beans which light roast on capacity 30 kg/hour was Rp362.9. Key words : Coffee roasted, Robusta, disk mill, mechanical grinder, size reduction.

  6. Micro- and Nano-vesicles from First Trimester Human Placentae Carry Flt-1 and Levels Are Increased in Severe Preeclampsia

    Directory of Open Access Journals (Sweden)

    Mancy Tong

    2017-07-01

    Full Text Available Background/objectivesPreeclampsia is a life-threatening hypertensive disease affecting 3–5% of pregnancies. While the pathogenesis of preeclampsia remains unclear, it is known that placenta-derived factors trigger the disease by activating maternal endothelial cells prior to the onset of clinical symptoms. Extracellular vesicles (EVs of different sizes extruded by the placenta may be one factor. The truncated/secreted form of Flt-1 (sFlt-1 has also been implicated in the pathogenesis of preeclampsia. We investigated whether placental EV production is altered in preeclampsia such that they induce endothelial cell activation, and whether (sFlt-1 is involved.MethodsMacro-, micro-, and nano-vesicles were collected from normal and preeclamptic (PE placental explants, and separated by differential centrifugation. The number and size of micro- and nano-vesicles was measured by nanoparticle tracking analysis and their ability to activate endothelial cells was quantified by endothelial cell intercellular adhesion molecule 1 expression and monocyte adhesion. The levels of Flt-1 were measured by western blots and ELISA.ResultsPE placentae extruded significantly more micro- and nano-vesicles than control placentae and the extruded micro-vesicles were larger than those from control placentae. Micro- and nano-vesicles from both first trimester and term human placentae carried Flt-1 and levels were significantly increased in EVs from severe, but not mild, PE compared to normotensive placentae. All fractions of EVs from PE placentae activated endothelial cells, and for micro- and nano-vesicles, activation was reduced in the presence of exogenous vascular endothelial growth factor (VEGF, a Flt-1 neutralizing antibody, or by pre-treatment with VEGF. While EV-bound VEGF constituted over 20% of the total detected VEGF secreted by PE and normotensive placentae, EV-bound Flt-1 did not significantly contribute to the total level of sFlt-1/Flt-1 released by human

  7. Dietary red palm oil supplementation reduces myocardial infarct size in an isolated perfused rat heart model

    Directory of Open Access Journals (Sweden)

    Esterhuyse Adriaan J

    2010-06-01

    Full Text Available Abstract Background and Aims Recent studies have shown that dietary red palm oil (RPO supplementation improves functional recovery following ischaemia/reperfusion in isolated hearts. The main aim of this study was to investigate the effects of dietary RPO supplementation on myocardial infarct size after ischaemia/reperfusion injury. The effects of dietary RPO supplementation on matrix metalloproteinase-2 (MMP2 activation and PKB/Akt phosphorylation were also investigated. Materials and methods Male Wistar rats were divided into three groups and fed a standard rat chow diet (SRC, a SRC supplemented with RPO, or a SRC supplemented with sunflower oil (SFO, for a five week period, respectively. After the feeding period, hearts were excised and perfused on a Langendorff perfusion apparatus. Hearts were subjected to thirty minutes of normothermic global ischaemia and two hours of reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. Coronary effluent was collected for the first ten minutes of reperfusion in order to measure MMP2 activity by gelatin zymography. Results Dietary RPO-supplementation decreased myocardial infarct size significantly when compared to the SRC-group and the SFO-supplemented group (9.1 ± 1.0% versus 30.2 ± 3.9% and 27.1 ± 2.4% respectively. Both dietary RPO- and SFO-supplementation were able to decrease MMP2 activity when compared to the SRC fed group. PKB/Akt phosphorylation (Thr 308 was found to be significantly higher in the dietary RPO supplemented group when compared to the SFO supplemented group at 10 minutes into reperfusion. There was, however, no significant changes observed in ERK phosphorylation. Conclusions Dietary RPO-supplementation was found to be more effective than SFO-supplementation in reducing myocardial infarct size after ischaemia/reperfusion injury. Both dietary RPO and SFO were able to reduce MMP2 activity, which suggests that MMP2 activity does not play a major role in

  8. Methotrexate carried in lipid core nanoparticles reduces myocardial infarction size and improves cardiac function in rats

    Science.gov (United States)

    Maranhão, Raul C; Guido, Maria C; de Lima, Aline D; Tavares, Elaine R; Marques, Alyne F; Tavares de Melo, Marcelo D; Nicolau, Jose C; Salemi, Vera MC; Kalil-Filho, Roberto

    2017-01-01

    Purpose Acute myocardial infarction (MI) is accompanied by myocardial inflammation, fibrosis, and ventricular remodeling that, when excessive or not properly regulated, may lead to heart failure. Previously, lipid core nanoparticles (LDE) used as carriers of the anti-inflammatory drug methotrexate (MTX) produced an 80-fold increase in the cell uptake of MTX. LDE-MTX treatment reduced vessel inflammation and atheromatous lesions induced in rabbits by cholesterol feeding. The aim of the study was to investigate the effects of LDE-MTX on rats with MI, compared with commercial MTX treatment. Materials and methods Thirty-eight Wistar rats underwent left coronary artery ligation and were treated with LDE-MTX, or with MTX (1 mg/kg intraperitoneally, once/week, starting 24 hours after surgery) or with LDE without drug (MI-controls). A sham-surgery group (n=12) was also included. Echocardiography was performed 24 hours and 6 weeks after surgery. The animals were euthanized and their hearts were analyzed for morphometry, protein expression, and confocal microscopy. Results LDE-MTX treatment achieved a 40% improvement in left ventricular (LV) systolic function and reduced cardiac dilation and LV mass, as shown by echocardiography. LDE-MTX reduced the infarction size, myocyte hypertrophy and necrosis, number of inflammatory cells, and myocardial fibrosis, as shown by morphometric analysis. LDE-MTX increased antioxidant enzymes; decreased apoptosis, macrophages, reactive oxygen species production; and tissue hypoxia in non-infarcted myocardium. LDE-MTX increased adenosine bioavailability in the LV by increasing adenosine receptors and modulating adenosine catabolic enzymes. LDE-MTX increased the expression of myocardial vascular endothelium growth factor (VEGF) associated with adenosine release; this correlated not only with an increase in angiogenesis, but also with other parameters improved by LDE-MTX, suggesting that VEGF increase played an important role in the beneficial

  9. Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs

    DEFF Research Database (Denmark)

    Yuan, Xiao Long; Zhang, Zhe; Pan, Rong Yang

    2017-01-01

    sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110-220 bp fragment size appeared to be deeper than in the 40-110 bp and 40...

  10. ISEV position paper: extracellular vesicle RNA analysis and bioinformatics

    Directory of Open Access Journals (Sweden)

    Andrew F. Hill

    2013-12-01

    Full Text Available Extracellular vesicles (EVs are the collective term for the various vesicles that are released by cells into the extracellular space. Such vesicles include exosomes and microvesicles, which vary by their size and/or protein and genetic cargo. With the discovery that EVs contain genetic material in the form of RNA (evRNA has come the increased interest in these vesicles for their potential use as sources of disease biomarkers and potential therapeutic agents. Rapid developments in the availability of deep sequencing technologies have enabled the study of EV-related RNA in detail. In October 2012, the International Society for Extracellular Vesicles (ISEV held a workshop on “evRNA analysis and bioinformatics.” Here, we report the conclusions of one of the roundtable discussions where we discussed evRNA analysis technologies and provide some guidelines to researchers in the field to consider when performing such analysis.

  11. Functionally polymerized surfactant vesicles: synthesis and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Tundo, P.; Kippenberger, D.J.; Klahn, P.L.; Prieto, N.E.; Fendler, J.H.

    1982-01-27

    Bis(2-(10-undecenoyloxycarbony bromide, bis(2-(10-undecenoyloxycarbony (2-hydroxyethyl)methylammonium bromide, bis(2-(10-undecenoyloxycarbony acid, bis(2-(10-undecenoyloxycarbony allylbis(2-dodecanoyloxycarbon bromide, and dimethyl-n-hexadecyl (10-(p-vin decyl)ammonium bromide have been synthesized. The predominantly single compartment bilayer vesicles formed from these surfactants could be polymerized either by exposure to ultraviolet irradiation or by the use of azoisobutyronitrile as an initiator. The presence of vesicles (unpolymerized and polymeric) has been demonstrated by electron micrography, H/sup 1/ NMR, gel filtration, phase transition, turbidity changes, substrate entrapment, and permeability. Polymerized vesicles are considerably more stable and less permeable and have reduced rates of turbidity changes compared to their unpolymerized counterparts. 19 references.

  12. Simplified microenvironments and reduced cell culture size influence the cell differentiation outcome in cellular microarrays.

    Science.gov (United States)

    Rodríguez-Seguí, Santiago A; Ortuño, María José; Ventura, Francesc; Martínez, Elena; Samitier, Josep

    2013-01-01

    Cellular microarrays present a promising tool for multiplex evaluation of the signalling effect of substrate-immobilized factors on cellular differentiation. In this paper, we compare the early myoblast-to-osteoblast cell commitment steps in response to a growth factor stimulus using standard well plate differentiation assays or cellular microarrays. Our results show that restraints on the cell culture size, inherent to cellular microarrays, impair the differentiation outcome. Also, while cells growing on spots with immobilised BMP-2 are early biased towards the osteoblast fate, longer periods of cell culturing in the microarrays result in cell proliferation and blockage of osteoblast differentiation. The results presented here raise concerns about the efficiency of cell differentiation when the cell culture dimensions are reduced to a simplified microspot environment. Also, these results suggest that further efforts should be devoted to increasing the complexity of the microspots composition, aiming to replace signalling cues missing in this system.

  13. Cleavage by RNase P of gene N mRNA reduces bacteriophage lambda burst size.

    Science.gov (United States)

    Li, Y; Altman, S

    1996-03-01

    RNase P, an enzyme essential for tRNA biosynthesis, can be directed to cleave any RNA when the target RNA is in a complex with a short, complementary oligonucleotide called an external guide sequence (EGS). RNase P from Escherichia coli can cleave phage lambda N mRNA in vitro or in vivo when the mRNA is in a complex with an EGS. The EGS can either be separate from or covalently linked to M1 RNA, the catalytic RNA subunit of RNase P. The requirement for Mg2+ in the reaction in vitro is lower when the EGS is covalently linked to M1 RNA. Substrates made of DNA can also be cleaved by RNase P in vitro in complexes with RNA EGSs. When either kind of EGS construct is used in vivo, burst size of phage lambda is reduced by > or = 40%. Reduction in burst size depends on efficient expression of the EGS constructs. The product of phage lambda gene N appears to function in a stoichiometric fashion.

  14. Slow Sedimentation and Deformability of Charged Lipid Vesicles

    Science.gov (United States)

    Rey Suárez, Iván; Leidy, Chad; Téllez, Gabriel; Gay, Guillaume; Gonzalez-Mancera, Andres

    2013-01-01

    The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity. PMID:23874582

  15. How pure are your vesicles?

    Science.gov (United States)

    Webber, Jason; Clayton, Aled

    2013-01-01

    We propose a straightforward method to estimate the purity of vesicle preparations by comparing the ratio of nano-vesicle counts to protein concentration, using tools such as the increasingly available NanoSight platform and a colorimetric protein assay such as the BCA-assay. Such an approach is simple enough to apply to every vesicle preparation within a given laboratory, assisting researchers as a routine quality control step. Also, the approach may aid in comparing/standardising vesicle purity across diverse studies, and may be of particular importance in evaluating vesicular biomarkers. We herein propose some criteria to aid in the definition of pure vesicles. PMID:24009896

  16. Lipid vesicle shape analysis from populations using light video microscopy and computer vision.

    Directory of Open Access Journals (Sweden)

    Jernej Zupanc

    Full Text Available We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1-50 µm in diameter. For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their projected diameters and isoperimetric quotients (measure of contour roundness. This process enables a comparison of samples from the same population over time, or the comparison of a treated population to a control. Although vesicles in suspensions are heterogeneous in sizes and shapes and have distinctively non-homogeneous distribution throughout the suspension, this method allows for the capture and analysis of repeatable vesicle samples that are representative of the population inspected.

  17. Restaurant owners' perspectives on a voluntary program to recognize restaurants for offering reduced-size portions, Los Angeles County, 2012.

    Science.gov (United States)

    Gase, Lauren; Dunning, Lauren; Kuo, Tony; Simon, Paul; Fielding, Jonathan E

    2014-03-20

    Reducing the portion size of food and beverages served at restaurants has emerged as a strategy for addressing the obesity epidemic; however, barriers and facilitators to achieving this goal are not well characterized. In fall 2012, the Los Angeles County Department of Public Health conducted semistructured interviews with restaurant owners to better understand contextual factors that may impede or facilitate participation in a voluntary program to recognize restaurants for offering reduced-size portions. Interviews were completed with 18 restaurant owners (representing nearly 350 restaurants). Analyses of qualitative data revealed 6 themes related to portion size: 1) perceived customer demand is central to menu planning; 2) multiple portion sizes are already being offered for at least some food items; 3) numerous logistical barriers exist for offering reduced-size portions; 4) restaurant owners have concerns about potential revenue losses from offering reduced-size portions; 5) healthful eating is the responsibility of the customer; and 6) a few owners want to be socially responsible industry leaders. A program to recognize restaurants for offering reduced-size portions may be a feasible approach in Los Angeles County. These findings may have applications for jurisdictions interested in engaging restaurants as partners in reducing the obesity epidemic.

  18. Restaurant Owners’ Perspectives on a Voluntary Program to Recognize Restaurants for Offering Reduced-Size Portions, Los Angeles County, 2012

    Science.gov (United States)

    Dunning, Lauren; Kuo, Tony; Simon, Paul; Fielding, Jonathan E.

    2014-01-01

    Introduction Reducing the portion size of food and beverages served at restaurants has emerged as a strategy for addressing the obesity epidemic; however, barriers and facilitators to achieving this goal are not well characterized. Methods In fall 2012, the Los Angeles County Department of Public Health conducted semistructured interviews with restaurant owners to better understand contextual factors that may impede or facilitate participation in a voluntary program to recognize restaurants for offering reduced-size portions. Results Interviews were completed with 18 restaurant owners (representing nearly 350 restaurants). Analyses of qualitative data revealed 6 themes related to portion size: 1) perceived customer demand is central to menu planning; 2) multiple portion sizes are already being offered for at least some food items; 3) numerous logistical barriers exist for offering reduced-size portions; 4) restaurant owners have concerns about potential revenue losses from offering reduced-size portions; 5) healthful eating is the responsibility of the customer; and 6) a few owners want to be socially responsible industry leaders. Conclusion A program to recognize restaurants for offering reduced-size portions may be a feasible approach in Los Angeles County. These findings may have applications for jurisdictions interested in engaging restaurants as partners in reducing the obesity epidemic. PMID:24650622

  19. Label-free tracking of single extracellular vesicles in a nano-fluidic optical fiber (Conference Presentation)

    Science.gov (United States)

    van der Pol, Edwin; Weidlich, Stefan; Lahini, Yoav; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk; Schmidt, Markus A.; Faez, Sanli; van Leeuwen, Ton G.

    2016-03-01

    Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter vesicles remains challenging. Thus far, vesicles vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (nvesicles vesicle-based clinical applications.

  20. Chronic Metformin Treatment is Associated with Reduced Myocardial Infarct Size in Diabetic Patients with ST-segment Elevation Myocardial Infarction

    NARCIS (Netherlands)

    Lexis, Chris P. H.; Wieringa, Wouter G.; Hiemstra, Bart; van Deursen, Vincent M.; Lipsic, Erik; van der Harst, Pim; van Veldhuisen, Dirk J.; van der Horst, Iwan C. C.

    Increased myocardial infarct (MI) size is associated with higher risk of developing left ventricular dysfunction, heart failure and mortality. Experimental studies have suggested that metformin treatment reduces MI size after induced ischaemia but human data is lacking. We aimed to investigate the

  1. Low-resolution simulations of vesicle suspensions in 2D

    Science.gov (United States)

    Kabacaoğlu, Gökberk; Quaife, Bryan; Biros, George

    2018-03-01

    Vesicle suspensions appear in many biological and industrial applications. These suspensions are characterized by rich and complex dynamics of vesicles due to their interaction with the bulk fluid, and their large deformations and nonlinear elastic properties. Many existing state-of-the-art numerical schemes can resolve such complex vesicle flows. However, even when using provably optimal algorithms, these simulations can be computationally expensive, especially for suspensions with a large number of vesicles. These high computational costs can limit the use of simulations for parameter exploration, optimization, or uncertainty quantification. One way to reduce the cost is to use low-resolution discretizations in space and time. However, it is well-known that simply reducing the resolution results in vesicle collisions, numerical instabilities, and often in erroneous results. In this paper, we investigate the effect of a number of algorithmic empirical fixes (which are commonly used by many groups) in an attempt to make low-resolution simulations more stable and more predictive. Based on our empirical studies for a number of flow configurations, we propose a scheme that attempts to integrate these fixes in a systematic way. This low-resolution scheme is an extension of our previous work [51,53]. Our low-resolution correction algorithms (LRCA) include anti-aliasing and membrane reparametrization for avoiding spurious oscillations in vesicles' membranes, adaptive time stepping and a repulsion force for handling vesicle collisions and, correction of vesicles' area and arc-length for maintaining physical vesicle shapes. We perform a systematic error analysis by comparing the low-resolution simulations of dilute and dense suspensions with their high-fidelity, fully resolved, counterparts. We observe that the LRCA enables both efficient and statistically accurate low-resolution simulations of vesicle suspensions, while it can be 10× to 100× faster.

  2. Losartan activates sirtuin 1 in rat reduced-size orthotopic liver transplantation.

    Science.gov (United States)

    Pantazi, Eirini; Bejaoui, Mohamed; Zaouali, Mohamed Amine; Folch-Puy, Emma; Pinto Rolo, Anabela; Panisello, Arnau; Palmeira, Carlos Marques; Roselló-Catafau, Joan

    2015-07-14

    To investigate a possible association between losartan and sirtuin 1 (SIRT1) in reduced-size orthotopic liver transplantation (ROLT) in rats. Livers of male Sprague-Dawley rats (200-250 g) were preserved in University of Wisconsin preservation solution for 1 h at 4 °C prior to ROLT. In an additional group, an antagonist of angiotensin II type 1 receptor (AT1R), losartan, was orally administered (5 mg/kg) 24 h and 1 h before the surgical procedure to both the donors and the recipients. Transaminase (as an indicator of liver injury), SIRT1 activity, and nicotinamide adenine dinucleotide (NAD(+), a co-factor necessary for SIRT1 activity) levels were determined by biochemical methods. Protein expression of SIRT1, acetylated FoxO1 (ac-FoxO1), NAMPT (the precursor of NAD+), heat shock proteins (HSP70, HO-1) expression, endoplasmic reticulum stress (GRP78, IRE1α, p-eIF2) and apoptosis (caspase 12 and caspase 3) parameters were determined by Western blot. Possible alterations in protein expression of mitogen activated protein kinases (MAPK), such as p-p38 and p-ERK, were also evaluated. Furthermore, the SIRT3 protein expression and mRNA levels were examined. The present study demonstrated that losartan administration led to diminished liver injury when compared to ROLT group, as evidenced by the significant decreases in alanine aminotransferase (358.3 ± 133.44 vs 206 ± 33.61, P losartan was associated with enhanced SIRT1 protein expression and activity (5.27 ± 0.32 vs 6.08 ± 0.30, P Losartan treatment also provoked significant attenuation of endoplasmic reticulum stress parameters (GRP78, IRE1α, p-eIF2) which was consistent with reduced levels of both caspase 12 and caspase 3. Furthermore, losartan administration stimulated HSP70 protein expression and attenuated HO-1 expression. However, no changes were observed in protein or mRNA expression of SIRT3. Finally, the protein expression pattern of p-ERK and p-p38 were not altered upon losartan administration. The

  3. Vitrification of Germinal Vesicle Stage Oocytes

    OpenAIRE

    ABE, Yasuyuki; AONO, Nobuya; Hara, Kenshiro; Matsumoto, Hiromichi; BAKHTIYARI, Mehrdad; Sasada, Hiroshi; Sato, Eimei

    2004-01-01

    In order to cryopreserve germinal vesicle (GV) stage oocytes, we first need to develop a novel container for keeping large quantities of GV oocytes, because of collecting them as cumulus oocytes complexes (COCs) that have bigger size and larger volume than oocytes themselves, and second modify a protocol for optimizing vitrification of them. In this mini-review, we describe our recent progress for attaining these objectives. When 65 bovine COCs having GV oocytes could be placed on a sheet of ...

  4. Intra- and trans-generational costs of reduced female body size caused by food limitation early in life in mites.

    Directory of Open Access Journals (Sweden)

    Andreas Walzer

    Full Text Available Food limitation early in life may be compensated for by developmental plasticity resulting in accelerated development enhancing survival at the expense of small adult body size. However and especially for females in non-matching maternal and offspring environments, being smaller than the standard may incur considerable intra- and trans-generational costs.Here, we evaluated the costs of small female body size induced by food limitation early in life in the sexually size-dimorphic predatory mite Phytoseiulus persimilis. Females are larger than males. These predators are adapted to exploit ephemeral spider mite prey patches. The intra- and trans-generational effects of small maternal body size manifested in lower maternal survival probabilities, decreased attractiveness for males, and a reduced number and size of eggs compared to standard-sized females. The trans-generational effects of small maternal body size were sex-specific with small mothers producing small daughters but standard-sized sons.Small female body size apparently intensified the well-known costs of sexual activity because mortality of small but not standard-sized females mainly occurred shortly after mating. The disadvantages of small females in mating and egg production may be generally explained by size-associated morphological and physiological constraints. Additionally, size-assortative mate preferences of standard-sized mates may have rendered small females disproportionally unattractive mating partners. We argue that the sex-specific trans-generational effects were due to sexual size dimorphism - females are the larger sex and thus more strongly affected by maternal stress than the smaller males - and to sexually selected lower plasticity of male body size.

  5. Invasive surgery reduces infarct size and preserves cardiac function in a porcine model of myocardial infarction.

    Science.gov (United States)

    van Hout, Gerardus P J; Teuben, Michel P J; Heeres, Marjolein; de Maat, Steven; de Jong, Renate; Maas, Coen; Kouwenberg, Lisanne H J A; Koenderman, Leo; van Solinge, Wouter W; de Jager, Saskia C A; Pasterkamp, Gerard; Hoefer, Imo E

    2015-11-01

    Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of the experimental procedures may influence outcome and thus clinical applicability. The aim of this study was to investigate if invasive surgery, as in the common open chest MI model affects IS and cardiac function. Twenty female landrace pigs were subjected to MI by transluminal balloon occlusion. In 10 of 20 pigs, balloon occlusion was preceded by invasive surgery (medial sternotomy). After 72 hrs, pigs were subjected to echocardiography and Evans blue/triphenyl tetrazoliumchloride double staining to determine IS and area at risk. Quantification of IS showed a significant IS reduction in the open chest group compared to the closed chest group (IS versus area at risk: 50.9 ± 5.4% versus 69.9 ± 3.4%, P = 0.007). End systolic LV volume and LV ejection fraction measured by echocardiography at follow-up differed significantly between both groups (51 ± 5 ml versus 65 ± 3 ml, P = 0.033; 47.5 ± 2.6% versus 38.8 ± 1.2%, P = 0.005). The inflammatory response in the damaged myocardium did not differ between groups. This study indicates that invasive surgery reduces IS and preserves cardiac function in a porcine MI model. Future studies need to elucidate the effect of infarct induction technique on the efficacy of pharmacological therapies in large animal cardioprotection studies. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  6. The toolbox of vesicle sidedness determination

    NARCIS (Netherlands)

    Meszaros, Peter; Hoekstra, Dick; Kok, Jan Willem

    2012-01-01

    Vesicles prepared from cellular plasma membranes are widely used in science for different purposes. The outer membrane leaflet differs from the inner membrane leaflet of the vesicle, and during vesicle preparation procedures two types of vesicles will be generated: right-side-out vesicles, of which

  7. Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus subtilis are Disrupted by the Lipopeptide Surfactin

    Science.gov (United States)

    Brown, Lisa; Kessler, Anne; Cabezas-Sanchez, Pablo; Luque-Garcia, Jose L.; Casadevall, Arturo

    2014-01-01

    Summary Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harboring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin. PMID:24826903

  8. Influence of reducing agents and surfactants on size and shape of silver fine powder particles

    Directory of Open Access Journals (Sweden)

    Stevan P. Dimitrijević

    2014-07-01

    Full Text Available Silver fine powder with different shapes and sizes were prepared by chemical reduction and characterized by scanning electron microscope. In this paper was presented the method for the preparation of the fine Ag powder with particles size smaller than 2.5 µm with suitability for the mass-production scale. Reduction was performed from nitrate solution directly by vigorous stirring at room temperature by three different reduction agents, with and without presence of two dispersants. Scanning electron microscopy revealed the preferred size of the particles obtained in all experiments with aim of the protecting agent. Larger particles and wider size distribution were obtained without surfactants although with average size of about 1 µm and small quantity of larger clusters of primary particles that is out of the fine powder classification. High purity, 99.999%, of silver was obtained in every experiment.

  9. Successful Transplantation of Reduced Sized Rat Alcoholic Fatty Livers Made Possible by Mobilization of Host Stem Cells

    Science.gov (United States)

    Hisada, Masayuki; Ota, Yoshihiro; Zhang, Xiuying; Cameron, Andrew M; Gao, Bin; Montgomery, Robert A; Williams, George Melville; Sun, Zhaoli

    2015-01-01

    Livers from Lewis rats fed with 7% alcohol for 5 weeks were used for transplantation. Reduced sized (50%) livers or whole livers were transplanted into normal DA recipients, which, in this strain combination, survive indefinitely when the donor has not been fed alcohol. However, none of the rats survived a whole fatty liver transplant while six of seven recipients of reduced sized alcoholic liver grafts survived long term. SDF-1 and HGF were significantly increased in reduced size liver grafts compared to whole liver grafts. Lineage-negative Thy-1+CXCR4+CD133+ stem cells were significantly increased in the peripheral blood and in allografts after reduced size fatty liver transplantation. In contrast, there were meager increases in cells reactive with anti Thy-1, CXCR4 and CD133 in peripheral blood and allografts in whole alcoholic liver recipients. The provision of plerixafor, a stem cell mobilizer, salvaged 5 of 10 whole fatty liver grafts. Conversely, blocking SDF-1 activity with neutralizing antibodies diminished stem cell recruitment and four of five reduced sized fatty liver recipients died. Thus chemokine insuficiency was associated with transplant failure of whole grafts which was overcome by the increased regenerative requirements promoted by the small grafts and mediated by SDF-1 resulting in stem cell influx. PMID:22994609

  10. The SNARE protein vti1a functions in dense-core vesicle biogenesis

    DEFF Research Database (Denmark)

    Walter, Alexander M; Kurps, Julia; de Wit, Heidi

    2014-01-01

    The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially...... overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion...... reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days...

  11. Effect of Counter Electrode in Electroformation of Giant Vesicles

    Directory of Open Access Journals (Sweden)

    Shuuhei Oana

    2011-11-01

    Full Text Available Electroformation of cell-sized lipid membrane vesicles (giant vesicles, GVs, from egg yolk phosphatidylcholine, was examined varying the shape of the counter electrode. Instead of a planar ITO (indium tin oxide electrode commonly used, platinum wire mesh was employed as a counter electrode facing lipid deposit on a planar formation electrode. The modification did not significantly alter GV formation, and many GVs of 30–50 µm, some as large as 100 µm, formed as with the standard setup, indicating that a counter electrode does not have to be a complete plane. When the counter electrode was reduced to a set of two parallel platinum wires, GV formation deteriorated. Some GVs formed, but only in close proximity to the counter electrode. Lower electric voltage with this setup no longer yielded GVs. Instead, a large onion-like multilamellar structure was observed. The deteriorated GV formation and the formation of a multilamellar structure seemed to indicate the weakened effect of the electric field on lipid deposit due to insufficient coverage with a small counter electrode. Irregular membranous objects formed by spontaneous swelling of lipid without electric voltage gradually turned into multilamellar structure upon following application of voltage. No particular enhancement of GV formation was observed when lipid deposit on a wire formation electrode was used in combination with a large planar counter electrode.

  12. Treatment with the gap junction modifier rotigaptide (ZP123) reduces infarct size in rats with chronic myocardial infarction

    DEFF Research Database (Denmark)

    Haugan, Ketil; Marcussen, Niels; Kjølbye, Anne Louise

    2006-01-01

    Treatment with non-selective drugs (eg, long-chain alcohols, halothane) that reduce gap junction intercellular communication (GJIC) is associated with reduced infarct size after myocardial infarction (MI). Therefore, it has been suggested that gap junction intercellular communication stimulating...... compounds may increase infarct size. The antiarrhythmic peptide analogue rotigaptide (ZP123) increases cardiac gap junction intercellular communication and the purpose of the present study was to examine the effects of rotigaptide treatment on infarct size. Myocardial infarction was induced in male rats...... by ligation of the left anterior descending artery (LAD). Rats (n = 156) were treated with rotigaptide at three dose levels or vehicle from the onset of ischemia and for 3 weeks following LAD occlusion. Infarct size was determined using histomorphometry after 3 weeks treatment. Rotigaptide treatment producing...

  13. Concurrent imaging of synaptic vesicle recycling and calcium dynamics.

    Directory of Open Access Journals (Sweden)

    Haiyan eLi

    2011-11-01

    Full Text Available Synaptic transmission involves the calcium-dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2, and a presynaptically-localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3 with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Re-acidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real-time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

  14. Preeclampsia and Extracellular Vesicles.

    Science.gov (United States)

    Gilani, Sarwat I; Weissgerber, Tracey L; Garovic, Vesna D; Jayachandran, Muthuvel

    2016-09-01

    Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

  15. Reduced glomerular size- and charge-selectivity in clinically healthy individuals with microalbuminuria

    DEFF Research Database (Denmark)

    Jensen, J S; Borch-Johnsen, K; Deckert, T

    1995-01-01

    The pathophysiologic mechanism behind microalbuminuria, a potential atherosclerotic risk factor, was explored by measuring fractional clearances of four endogenous plasma proteins of different size and electric charge (albumin, beta 2-microglobulin, immunoglobulin G, and immunoglobulin G4). Twenty...

  16. Invasive surgery reduces infarct size and preserves cardiac function in a porcine model of myocardial infarction

    NARCIS (Netherlands)

    G.P.J. van Hout (G. P J); M.P.J. Teuben (Michel P.J.); M. Heeres (Marjolein); S. de Maat (Steven); R. Jong (Rosa); C. Maas (Coen); L.H.J.A. Kouwenberg (Lisanne H.J.A.); L. Koenderman (Leo); W.W. van Solinge (Wouter W.); S.C.A. de Jager (Saskia); G. Pasterkamp (Gerard); I.E. Hoefer (Imo)

    2015-01-01

    textabstract& Sons Ltd and Foundation for Cellular and Molecular Medicine. Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting

  17. Invasive surgery reduces infarct size and preserves cardiac function in a porcine model of myocardial infarction

    NARCIS (Netherlands)

    van Hout, Gerardus P J; Teuben, Michel P J; Heeres, Marjolein; de Maat, Steven; de Jong, Renate; Maas, Coen; Kouwenberg, Lisanne H J A; Koenderman, Leo; van Solinge, Wouter W; de Jager, Saskia C A; Pasterkamp, Gerard; Höfer, IE

    2015-01-01

    Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of

  18. Theory of dielectric response of charged-bilayer-vesicle solutions

    Science.gov (United States)

    Lu, C.-Y. D.

    1996-10-01

    The dielectric response is calculated for a solution containing charged bilayer vesicles and simple electrolyte. The solution is assumed to contain a high salt concentration so that the Debye screening length is small compared to the size of the vesicles. The presence of two (electric) double layers, one on each side of the bilayer, gives low-frequency salt relaxations (kHz for 1 μm vesicles) that explain the experimentally observed α relaxations which are known to appear only for charged vesicles. The double layers also modify the high-frequency β relaxations which have been previously modeled by using the Maxwell-Wagner theory. The calculation method can be easily extended to other bilayer geometries.

  19. Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.

    Science.gov (United States)

    Sotillo, Javier; Pearson, Mark; Potriquet, Jeremy; Becker, Luke; Pickering, Darren; Mulvenna, Jason; Loukas, Alex

    2016-01-01

    Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host-schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Reduced size-independent mechanical properties of cortical bone in high-fat diet-induced obesity.

    Science.gov (United States)

    Ionova-Martin, S S; Do, S H; Barth, H D; Szadkowska, M; Porter, A E; Ager, J W; Ager, J W; Alliston, T; Vaisse, C; Ritchie, R O

    2010-01-01

    Overweight and obesity are rapidly expanding health problems in children and adolescents. Obesity is associated with greater bone mineral content that might be expected to protect against fracture, which has been observed in adults. Paradoxically, however, the incidence of bone fractures has been found to increase in overweight and obese children and adolescents. Prior studies have shown some reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent measures of mechanical properties, which are important to understand material behavior. To clarify the effects of HFD on the mechanical properties and microstructure of bone, femora from C57BL/6 mice fed either a HFD or standard laboratory chow (Chow) were evaluated for structural changes and tested for bending strength, bending stiffness and fracture toughness. Here, we find that in young, obese, high-fat fed mice, all geometric parameters of the femoral bone, except length, are increased, but strength, bending stiffness, and fracture toughness are all reduced. This increased bone size and reduced size-independent mechanical properties suggests that obesity leads to a general reduction in bone quality despite an increase in bone quantity; yield and maximum loads, however, remained unchanged, suggesting compensatory mechanisms. We conclude that diet-induced obesity increases bone size and reduces size-independent mechanical properties of cortical bone in mice. This study indicates that bone quantity and bone quality play important compensatory roles in determining fracture risk. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  1. Leukocytospermia and function of the seminal vesicles on seminal quality.

    Science.gov (United States)

    Gonzales, G F; Kortebani, G; Mazzolli, A B

    1992-05-01

    To determine possible relationships between number of leukocytes, function of seminal vesicles, and seminal quality. The study was carried out on men who consecutively attended an infertility clinic between June 1989 to June 1991. This study was conducted in a private immunological center for infertility, a tertiary care center, The Centro Immunológico-Sección Esterilidad y Reproducción. Semen samples from 280 infertility patients attending an Immunological Center for Infertility were analyzed. We evaluated the effect of leukocytospermia in the presence of normal or abnormal function of seminal vesicles on seminal quality. Sperm count, percent of motile sperm, and percent of sperm vitality were significantly reduced when both leukocytospermia and hypofunction of seminal vesicles were present (P less than 0.01). Leukocytospermic subjects with normal function of seminal vesicles showed similar seminal parameters to those nonleukocytspermics. The incidence of subjects with antisperm antibodies measured by direct immunobeads was significantly higher in leukocytospermic men with hypofunction of seminal vesicles. No differences in the incidence of antisperm antibodies with nonleukocytospermic samples were observed in those with both leukocytospermia and normal function of seminal vesicles. These data provide evidence that white blood cells were deleterious for seminal quality when seminal vesicles were also affected.

  2. Acarbose reduces myocardial infarct size by preventing postprandial hyperglycemia and hydroxyl radical production and opening mitochondrial KATP channels in rabbits.

    Science.gov (United States)

    Minatoguchi, Shinya; Zhang, Zengi; Bao, Narentuoya; Kobayashi, Hiroyuki; Yasuda, Shinji; Iwasa, Masamitsu; Sumi, Syouhei; Kawamura, Itta; Yamada, Yoshihisa; Nishigaki, Kazuhiko; Takemura, Genzou; Fujiwara, Takako; Fujiwara, Hisayoshi

    2009-07-01

    Acarbose, an antidiabetic drug, is an alpha-glucosidase inhibitor that can inhibit glucose absorption in the intestine. A recent large-scale clinical trial, STOP-NIDDM, showed that acarbose reduces the risk of myocardial infarction. We examined whether acarbose reduces myocardial infarct size and investigated its mechanisms. Rabbits were fed with 1 of 2 diets in this study: normal chow, 30 mg acarbose per 100 g chow for 7 days. Rabbits were assigned randomly to 1 of 4 groups: control (n = 10), acarbose (n = 10), acarbose + 5HD (n = 10, intravenous 5 mg/kg of 5-hydroxydecanoate), and 5HD (n = 10, intravenous 5 mg/kg of 5HD). Rabbits then underwent 30 minutes of coronary occlusion followed by 48-hour reperfusion. Postprandial blood glucose levels were higher in the control group than in the acarbose group. The infarct size as a percentage of the left ventricular area at risk was reduced significantly in the acarbose (19.4% +/- 2.3%) compared with the control groups (42.8% +/- 5.4%). The infarct size-reducing effect of acarbose was abolished by 5HD (43.4% +/- 4.7%). Myocardial interstitial 2,5-dihydroxybenzoic acid levels, an indicator of hydroxyl radicals, increased during reperfusion after 30 minutes of ischemia, but this increase was inhibited in the acarbose group. This was reversed by 5HD. Acarbose reduces myocardial infarct size by opening mitochondrial KATP channels, which may be related to the prevention of postprandial hyperglycemia and hydroxyl radical production.

  3. An Informatics Based Approach to Reduce the Grain Size of Cast Hadfield Steel

    Science.gov (United States)

    Dey, Swati; Pathak, Shankha; Sheoran, Sumit; Kela, Damodar H.; Datta, Shubhabrata

    2016-04-01

    Materials Informatics concept using computational intelligence based approaches are employed to bring out the significant alloying additions to achieve grain refinement in cast Hadfield steel. Castings of Hadfield steels used for railway crossings, requires fine grained austenitic structure. Maintaining proper grain size of this component is very crucial in order to achieve the desired properties and service life. This work studies the important variables affecting the grain size of such steels which includes the compositional and processing variables. The computational findings and prior knowledge is used to design the alloy, which is subjected to a few trials to validate the findings.

  4. Community size and metabolic rates of psychrophilic sulfate-reducing bacteria in Arctic marine sediments

    DEFF Research Database (Denmark)

    Knoblauch, C.; Jørgensen, BB; Harder, J.

    1999-01-01

    of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than...

  5. Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients.

    Science.gov (United States)

    Bryzgunova, Olga E; Zaripov, Marat M; Skvortsova, Tatyana E; Lekchnov, Evgeny A; Grigor'eva, Alina E; Zaporozhchenko, Ivan A; Morozkin, Evgeny S; Ryabchikova, Elena I; Yurchenko, Yuri B; Voitsitskiy, Vladimir E; Laktionov, Pavel P

    2016-01-01

    Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90-95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

  6. Optimal foraging on perilous prey : risk of bill damage reduces optimal prey size in oystercatchers

    NARCIS (Netherlands)

    Rutten, AL; Oosterbeek, K; Ens, Bruno J.; Verhulst, S

    2006-01-01

    Intake rate maximization alone is not. always sufficient in explaining prey size selection in predators. For example, bivalve-feeding oystercatchers regularly select. smaller prey than expected if they aimed to maximize their intake rat-C. It has been proposed that. to these birds large prey are

  7. Optimal foraging on perilous prey: risk of bill damage reduces optimal prey size in oystercatchers

    NARCIS (Netherlands)

    Rutten, A.L.; Oosterbeek, K.H.; Ens, B.J.; Verhulst, S.

    2006-01-01

    Intake rate maximization alone is not always sufficient in explaining prey size selection in predators. For example, bivalve-feeding oystercatchers regularly select smaller prey than expected if they aimed to maximize their intake rate. It has been proposed that to these birds large prey are

  8. Buffer sizing to reduce interference and increase throughput of real-time stream processing applications

    NARCIS (Netherlands)

    Kurtin, Philip Sebastian; Geuns, S.J.; Hausmans, J.P.H.M.; Bekooij, Marco Jan Gerrit

    2015-01-01

    Existing temporal analysis and buffer sizing techniques for real-time stream processing applications ignore that FIFO buffers bound interference between tasks on the same processor. By considering this effect it can be shown that a reduction of buffer capacities can result in a higher throughput.

  9. Rapid short-duration hypothermia with cold saline and endovascular cooling before reperfusion reduces microvascular obstruction and myocardial infarct size

    Directory of Open Access Journals (Sweden)

    Heiberg Einar

    2008-04-01

    Full Text Available Abstract Background The aim of this study was to evaluate the combination of a rapid intravenous infusion of cold saline and endovascular hypothermia in a closed chest pig infarct model. Methods Pigs were randomized to pre-reperfusion hypothermia (n = 7, post-reperfusion hypothermia (n = 7 or normothermia (n = 5. A percutaneous coronary intervention balloon was inflated in the left anterior descending artery for 40 min. Hypothermia was started after 25 min of ischemia or immediately after reperfusion by infusion of 1000 ml of 4°C saline and endovascular hypothermia. Area at risk was evaluated by in vivo SPECT. Infarct size was evaluated by ex vivo MRI. Results Pre-reperfusion hypothermia reduced infarct size/area at risk by 43% (46 ± 8% compared to post-reperfusion hypothermia (80 ± 6%, p Conclusion Rapid hypothermia with cold saline and endovascular cooling before reperfusion reduces myocardial infarct size and microvascular obstruction. A novel finding is that hypothermia at the onset of reperfusion reduces microvascular obstruction without reducing myocardial infarct size. Intravenous administration of cold saline combined with endovascular hypothermia provides a method for a rapid induction of hypothermia suggesting a potential clinical application.

  10. Comparing two psychological interventions in reducing impulsive processes of eating behaviour: Effects on self-selected portion size

    NARCIS (Netherlands)

    Koningsbruggen, G.M. van; Veling, H.P.; Stroebe, W.; Aarts, H.A.G.

    2014-01-01

    Objective Palatable food, such as sweets, contains properties that automatically trigger the impulse to consume it even when people have goals or intentions to refrain from consuming such food. We compared the effectiveness of two interventions in reducing the portion size of palatable food that

  11. Comparing two psychological interventions in reducing impulsive processes of eating behaviour : Effects on self-selected portion size

    NARCIS (Netherlands)

    van Koningsbruggen, G.M.; Veling, H.P.; Stroebe, Wolfgang; Aarts, Henk

    2014-01-01

    ObjectivePalatable food, such as sweets, contains properties that automatically trigger the impulse to consume it even when people have goals or intentions to refrain from consuming such food. We compared the effectiveness of two interventions in reducing the portion size of palatable food that

  12. BIOREACTOR ECONOMICS, SIZE AND TIME OF OPERATION (BEST) COMPUTER SIMULATOR FOR DESIGNING SULFATE-REDUCING BACTERIA FIELD BIOREACTORS

    Science.gov (United States)

    BEST (bioreactor economics, size and time of operation) is an Excel™ spreadsheet-based model that is used in conjunction with the public domain geochemical modeling software, PHREEQCI. The BEST model is used in the design process of sulfate-reducing bacteria (SRB) field bioreacto...

  13. Selectively Reduced Posterior Corpus Callosum Size in a Population-Based Sample of Young Adults Born with Low Birth Weight

    DEFF Research Database (Denmark)

    Aukland, S M; Westerhausen, R; Plessen, K J

    2011-01-01

    BACKGROUND AND PURPOSE: Several studies suggest that VLBW is associated with a reduced CC size later in life. We aimed to clarify this in a prospective, controlled study of 19-year-olds, hypothesizing that those with LBWs had smaller subregions of CC than the age-matched controls, even after...

  14. Derivatives of a statically reduced stiffness matrix with respect to sizing variables. [for aircraft weight minimization with flutter constraints

    Science.gov (United States)

    Oconnell, R. F.; Hassig, H. J.; Radovcich, N. A.

    1976-01-01

    An expression is obtained for the first derivatives with respect to the sizing variables of a statically reduced stiffness matrix that is a nonlinear function of the sizing variables, where the unreduced stiffness matrix is a linear function of the sizing variables. An accepted procedure to reduce the number of degrees of freedom is to eliminate a number of nodal displacements from the degrees of freedom such that the accuracy of the flutter analysis is not significantly affected. In a typical optimization procedure with flutter constraints, the derivative of the stiffness matrix may be used in a form that contains the characteristic vector of the flutter matrix equation and the transpose of the characteristic vector of the adjoint flutter matrix equation corresponding to a particular solution of the flutter equation.

  15. Novel, selective EPO receptor ligands lacking erythropoietic activity reduce infarct size in acute myocardial infarction in rats.

    Science.gov (United States)

    Kiss, Krisztina; Csonka, Csaba; Pálóczi, János; Pipis, Judit; Görbe, Anikó; Kocsis, Gabriella F; Murlasits, Zsolt; Sárközy, Márta; Szűcs, Gergő; Holmes, Christopher P; Pan, Yijun; Bhandari, Ashok; Csont, Tamás; Shamloo, Mehrdad; Woodburn, Kathryn W; Ferdinandy, Péter; Bencsik, Péter

    2016-11-01

    Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100U/mL significantly decreased cell death compared to vehicle (33.8±2.3% vs. 40.3±1.5%, pInfarct size (IS) was measured by standard TTC staining. In study 1, 5000U/kg EPO reduced infarct size significantly compared to vehicle (45.3±4.8% vs. 59.8±4.5%, pinfarct size-reducing effect at 5μg/kg compared to the vehicle (44.4±5.7% vs. 65.9±2.7%, pinfarct size in studies 1-3 by approximately 35%. In study 4, AF43136 at 10mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle (39.4±5.9% vs. 58.1±5.4% and 45.9±2.4% vs. 63.8±4.1%, pinfarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    Science.gov (United States)

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge. PMID:27989272

  17. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

    Science.gov (United States)

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

  18. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Cecilia Lässer

    2016-12-01

    Full Text Available The International Society for Extracellular Vesicles (ISEV has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs. This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge.

  19. Measuring proteins with greater speed and resolution while reducing sample size

    OpenAIRE

    Hsieh, Vincent H.; Wyatt, Philip J.

    2017-01-01

    A multi-angle light scattering (MALS) system, combined with chromatographic separation, directly measures the absolute molar mass, size and concentration of the eluate species. The measurement of these crucial properties in solution is essential in basic macromolecular characterization and all research and production stages of bio-therapeutic products. We developed a new MALS methodology that has overcome the long-standing, stubborn barrier to microliter-scale peak volumes and achieved the hi...

  20. Reduced Particle size of plant material does not stimulate decomposition but affects the microbivorous microfauna

    DEFF Research Database (Denmark)

    Vestergaard, Peter; Rønn, Regin; Christensen, Søren

    2001-01-01

    in soils amended with the large pieces on nine out of 10 occasions. Microbial biomass measured as SIR was significantly higher in soils with maize than in those amended with barley, but no effect of particle size was observed (three-way ANOVA, P... material, but significantly higher numbers were found in soil with finely-ground maize than in soil with large pieces (two-way ANOVA, P... barley (three-way ANOVA, P

  1. Detection of platelet vesicles by flow cytometry.

    Science.gov (United States)

    Nolan, John P; Jones, Jennifer C

    2017-05-01

    The composition and function of platelet-derived extracellular vesicles (EVs) in health and in disease are a major topic of investigation in biomedical research. However, efforts to delineate specific molecular repertoires and roles for different types of EVs in the circulation are limited not only by the lack of flow cytometers capable of analyzing submicron- and nano-materials across the full size spectrum of plasma EVs, but also by the lack of standardized methods and reference materials that would permit inter-laboratory reproducibility for these analyses. In this review, we summarize the flow cytometry of EVs, with a focus on platelet vesicles in plasma. In addition to delineating the basic principles that govern what precautions must be considered when using flow cytometry for the analysis of platelet vesicles, we provide an overview for how to standardize, control, annotate, and report EV flow cytometry data reproducibly, while looking forward to a next generation of high sensitivity instruments for the analysis of EVs and other submicron biomaterials in the circulation.

  2. Asymmetric osmotic water permeation through a vesicle membrane

    Science.gov (United States)

    Su, Jiaye; Zhao, Yunzhen; Fang, Chang; Shi, Yue

    2017-05-01

    Understanding the water permeation through a cell membrane is of primary importance for biological activities and a key step to capture its shape transformation in salt solution. In this work, we reveal the dynamical behaviors of osmotically driven transport of water molecules across a vesicle membrane by molecular dynamics simulations. Of particular interest is that the water transport in and out of vesicles is highly distinguishable given the osmotic force are the same, suggesting an asymmetric osmotic transportation. This asymmetric phenomenon exists in a broad range of parameter space such as the salt concentration, temperature, and vesicle size and can be ascribed to the similar asymmetric potential energy of lipid-ion, lipid-water, lipid-solution, lipid-lipid, and the lipid-lipid energy fluctuation. Specifically, the water flux has a linear increase with the salt concentration, similar to the prediction by Nernst-Planck equation or Fick's first law. Furthermore, due to the Arrhenius relation between the membrane permeability and temperature, the water flux also exhibits excellent Arrhenius dependence on the temperature. Meanwhile, the water flux shows a linear increase with the vesicle surface area since the flux amount across a unit membrane area should be a constant. Finally, we also present the anonymous diffusion behaviors for the vesicle itself, where transitions from normal diffusion at short times to subdiffusion at long times are identified. Our results provide significant new physical insights for the osmotic water permeation through a vesicle membrane and are helpful for future experimental studies.

  3. Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3beta?

    LENUS (Irish Health Repository)

    Carew, Rosemarie M.

    2010-07-06

    Abstract Background Male Irs2-\\/- mice develop fatal type 2 diabetes at 13-14 weeks. Defects in neuronal proliferation, pituitary development and photoreceptor cell survival manifest in Irs2-\\/- mice. We identify retarded renal growth in male and female Irs2-\\/- mice, independent of diabetes. Results Kidney size and kidney:body weight ratio were reduced by approximately 20% in Irs2-\\/- mice at postnatal day 5 and was maintained in maturity. Reduced glomerular number but similar glomerular density was detected in Irs2-\\/- kidney compared to wild-type, suggesting intact global kidney structure. Analysis of insulin signalling revealed renal-specific upregulation of PKBβ\\/Akt2, hyperphosphorylation of GSK3β and concomitant accumulation of β-catenin in Irs2-\\/- kidney. Despite this, no significant upregulation of β-catenin targets was detected. Kidney-specific increases in Yes-associated protein (YAP), a key driver of organ size were also detected in the absence of Irs2. YAP phosphorylation on its inhibitory site Ser127 was also increased, with no change in the levels of YAP-regulated genes, suggesting that overall YAP activity was not increased in Irs2-\\/- kidney. Conclusions In summary, deletion of Irs2 causes reduced kidney size early in mouse development. Compensatory mechanisms such as increased β-catenin and YAP levels failed to overcome this developmental defect. These data point to Irs2 as an important novel mediator of kidney size.

  4. Adsorption and encapsulation of flexible polyelectrolytes in charged spherical vesicles

    Science.gov (United States)

    Shojaei, H. R.; Muthukumar, M.

    2017-06-01

    We present a theory of adsorption of flexible polyelectrolytes on the interior and exterior surfaces of a charged vesicle in an electrolyte solution. The criteria for adsorption and the density profiles of the adsorbed polymer chain are derived in terms of various characteristics of the polymer, vesicle, and medium, such as the charge density and length of the polymer, charge density and size of the vesicle, electrolyte concentration and dielectric constant of the medium. For adsorption inside the vesicle, the competition between the loss of conformational entropy and gain in adsorption energy results in two kinds of encapsulated states, depending on the strength of the polymer-vesicle interaction. By considering also the adsorption from outside the vesicle, we derive the entropic and energy contributions to the free energy change to transfer an adsorbed chain in the interior to an adsorbed chain on the exterior. In this paper, we have used the Wentzel-Kramers-Brillouin (WKB) method to solve the equation for the probability distribution function of the chain. The present WKB results are compared with the previous results based on variational methods. The WKB and variational results are in good agreement for both the interior and exterior states of adsorption, except in the zero-salt limit for adsorption in the exterior region. The adsorption criteria and density profiles for both the interior and exterior states are presented in terms of various experimentally controllable variables. Calculation of the dependencies of free energy change to transfer an adsorbed chain from the interior to the exterior surface on salt concentration and vesicle radius shows that the free energy penalty to expel a chain from a vesicle is only of the order of thermal energy.

  5. Emergent properties of extracellular vesicles: a holistic approach to decode the complexity of intercellular communication networks.

    Science.gov (United States)

    Gho, Yong Song; Lee, Changjin

    2017-06-27

    Shedding of nano-sized bilayered extracellular vesicles and extracellular vesicle-mediated intercellular communication are evolutionarily conserved biological processes. Communication between cells and the environment is an essential process in living organisms and dysregulation of intercellular communication leads to various diseases. Thus, systematic studies on extracellular vesicles, also known as exosomes, microvesicles, and outer membrane vesicles, are critical for a deeper understanding of intercellular communication networks that are crucial for decoding the exact causes of various difficult-to-cure diseases. Recent progress in this emerging field reveals that extracellular vesicles are endogenous carriers of specific subsets of proteins, mRNAs, miRNAs, and other bioactive materials, as well as play diverse pathophysiological roles. However, certain issues regarding diverse subtypes and the complex pathophysiological roles of extracellular vesicles are not yet clearly elucidated. In this review, we first briefly introduce the complexity of extracellular vesicles in terms of their vesicular cargos and protein-protein interaction networks, their diverse subtypes, and multifaceted pathophysiological functions. Then, we introduce the limitation of reductionist approaches in understanding the complexity of extracellular vesicles. We finally suggest that molecular systems biology approaches based on the concept of emergent properties are essential for a comprehensive understanding of the complex pathophysiological functions of heterogeneous extracellular vesicles, either at the single vesicle level or at a systems level as a whole.

  6. Hyperbranched polymer vesicles: from self-assembly, characterization, mechanisms, and properties to applications.

    Science.gov (United States)

    Jiang, Wenfeng; Zhou, Yongfeng; Yan, Deyue

    2015-06-21

    Vesicles, including lipid vesicles, surfactant vesicles, as well as polymer vesicles, have been extensively investigated over the past fifty years. Among them, polymer vesicles have attracted more and more attention because of their low permeability, superior stability and toughness, in addition to the numerous possibilities for tailoring physical, chemical and biological properties. Polymer vesicles are generally fabricated through the self-assembly of amphiphilic polymers with a linear architecture. Recently, as representative polymers with a highly branched three-dimensional architecture, hyperbranched polymers have also exhibited great potential for preparing vesicles. The resultant hyperbranched polymer vesicles, defined as branched-polymersomes (BPs), have shown unique properties, such as giant and easily tuned vesicle sizes, facile functionalization, a special formation mechanism, and appealing solution behaviours. In this tutorial review, ten years of advances in BPs have been summarized since their first discovery in the year 2004, including the syntheses of vesicle-forming hyperbranched polymers, self-assembly methods, self-assembly mechanisms, as well as the special properties. In addition, the cytomimetic, biomedical and other initiatory applications of BPs are also included.

  7. High-intensity training reduces intermittent hypoxia-induced ER stress and myocardial infarct size.

    Science.gov (United States)

    Bourdier, Guillaume; Flore, Patrice; Sanchez, Hervé; Pepin, Jean-Louis; Belaidi, Elise; Arnaud, Claire

    2016-01-15

    Chronic intermittent hypoxia (IH) is described as the major detrimental factor leading to cardiovascular morbimortality in obstructive sleep apnea (OSA) patients. OSA patients exhibit increased infarct size after a myocardial event, and previous animal studies have shown that chronic IH could be the main mechanism. Endoplasmic reticulum (ER) stress plays a major role in the pathophysiology of cardiovascular disease. High-intensity training (HIT) exerts beneficial effects on the cardiovascular system. Thus, we hypothesized that HIT could prevent IH-induced ER stress and the increase in infarct size. Male Wistar rats were exposed to 21 days of IH (21-5% fraction of inspired O2, 60-s cycle, 8 h/day) or normoxia. After 1 wk of IH alone, rats were submitted daily to both IH and HIT (2 × 24 min, 15-30m/min). Rat hearts were either rapidly frozen to evaluate ER stress by Western blot analysis or submitted to an ischemia-reperfusion protocol ex vivo (30 min of global ischemia/120 min of reperfusion). IH induced cardiac proapoptotic ER stress, characterized by increased expression of glucose-regulated protein kinase 78, phosphorylated protein kinase-like ER kinase, activating transcription factor 4, and C/EBP homologous protein. IH-induced myocardial apoptosis was confirmed by increased expression of cleaved caspase-3. These IH-associated proapoptotic alterations were associated with a significant increase in infarct size (35.4 ± 3.2% vs. 22.7 ± 1.7% of ventricles in IH + sedenary and normoxia + sedentary groups, respectively, P < 0.05). HIT prevented both the IH-induced proapoptotic ER stress and increased myocardial infarct size (28.8 ± 3.9% and 21.0 ± 5.1% in IH + HIT and normoxia + HIT groups, respectively, P = 0.28). In conclusion, these findings suggest that HIT could represent a preventive strategy to limit IH-induced myocardial ischemia-reperfusion damages in OSA patients. Copyright © 2016 the American Physiological Society.

  8. Selection for number of live piglets at five-days of age increased litter size and reduced mortality

    DEFF Research Database (Denmark)

    Nielsen, Bjarne; Madsen, Per; Henryon, Mark

    2012-01-01

    . The heritabilities of maternal effect on litter size were 0.079 and 0.095 in Landrace and Yorkshir e. The heritabilities of maternal effect on piglet-mortality rates were 0.069 and 0.082 in Landrace and Yorkshire. The genetic correlation between litter size and mortality rate were unfavourable; and the estimates......-netic gain has reduced the piglet mortality rate by 4 %-points in Landrace and Yorkshire from 2004 to 2010. The genetics gain was confirmed by decreased phenotypic annual mortality rates in the breeding and multiplier herds....

  9. Growth and instability of a phospholipid vesicle in a bath of fatty acids

    Science.gov (United States)

    Dervaux, J.; Noireaux, V.; Libchaber, A. J.

    2017-06-01

    Using a microfluidic trap, we study the behavior of individual phospholipid vesicles in contact with fatty acids. We show that spontaneous fatty acids insertion inside the bilayer is controlled by the vesicle size, osmotic pressure difference across the membrane and fatty acids concentration in the external bath. Depending on these parameters, vesicles can grow spherically or become unstable and fragment into several daughter vesicles. We establish the phase diagram for vesicle growth and we derive a simple thermodynamic model that reproduces the time evolution of the vesicle volume. Finally, we show that stable growth can be achieved on an artificial cell expressing a simple set of bacterial cytoskeletal proteins, paving the way toward artificial cell reproduction.

  10. Vesicles from Amphiphilic Dumbbells and Janus Dendrimers: Bioinspired Self-Assembled Structures for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Soraya Taabache

    2017-07-01

    Full Text Available The current review focuses on vesicles obtained from the self-assembly of two types of dendritic macromolecules, namely amphiphilic Janus dendrimers (forming dendrimersomes and amphiphilic dumbbells. In the first part, we will present some synthetic strategies and the various building blocks that can be used to obtain dendritic-based macromolecules, thereby showing their structural versatility. We put our focus on amphiphilic Janus dendrimers and amphiphilic dumbbells that form vesicles in water but we also encompass vesicles formed thereof in organic solvents. The second part of this review deals with the production methods of these vesicles at the nanoscale but also at the microscale. Furthermore, the influence of various parameters (intrinsic to the amphiphilic JD and extrinsic—from the environment on the type of vesicle formed will be discussed. In the third part, we will review the numerous biomedical applications of these vesicles of nano- or micron-size.

  11. Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

    DEFF Research Database (Denmark)

    Kristensen, Kasper; Henriksen, Jonas Rosager; Andresen, Thomas Lars

    2014-01-01

    that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar...

  12. Herniated disks unchanged over time: Size reduced after oxygen-ozone therapy.

    Science.gov (United States)

    Bonetti, Matteo; Zambello, Alessio; Leonardi, Marco; Princiotta, Ciro

    2016-08-01

    The spontaneous regression of disk herniation secondary to dehydration is a much-debated topic in medicine. Some physicians wonder whether surgical removal of the extruded nucleus pulposus is really necessary when the spontaneous disappearance of a herniated lumbar disk is a well-known phenomenon. Unfortunately, without spontaneous regression, chronic pain leads to progressive disability for which surgery seems to be the only solution. In recent years, several studies have demonstrated the utility of oxygen-ozone therapy in the treatment of disk herniation, resulting in disk shrinkage. This retrospective study evaluates the outcomes of a series of patients with a history of herniated disks neuroradiologically unchanged in size for over two years, treated with oxygen-ozone therapy at our center over the last 15 years. We treated 96 patients, 84 (87.5%) presenting low back pain complicated or not by chronic sciatica. No drug therapy had yielded significant benefits. A number of specialists had been consulted in two or more years resulting in several neuroradiological scans prior to the decision to undertake oxygen-ozone therapy. Our study documents how ozone therapy for slipped disks "unchanged over time" solved the problem, with disk disruption or a significant reduction in the size of the prolapsed disk material extruded into the spinal canal. © The Author(s) 2016.

  13. Herniated disks unchanged over time: Size reduced after oxygen–ozone therapy

    Science.gov (United States)

    Bonetti, Matteo; Zambello, Alessio; Princiotta, Ciro

    2016-01-01

    The spontaneous regression of disk herniation secondary to dehydration is a much-debated topic in medicine. Some physicians wonder whether surgical removal of the extruded nucleus pulposus is really necessary when the spontaneous disappearance of a herniated lumbar disk is a well-known phenomenon. Unfortunately, without spontaneous regression, chronic pain leads to progressive disability for which surgery seems to be the only solution. In recent years, several studies have demonstrated the utility of oxygen–ozone therapy in the treatment of disk herniation, resulting in disk shrinkage. This retrospective study evaluates the outcomes of a series of patients with a history of herniated disks neuroradiologically unchanged in size for over two years, treated with oxygen–ozone therapy at our center over the last 15 years. We treated 96 patients, 84 (87.5%) presenting low back pain complicated or not by chronic sciatica. No drug therapy had yielded significant benefits. A number of specialists had been consulted in two or more years resulting in several neuroradiological scans prior to the decision to undertake oxygen–ozone therapy. Our study documents how ozone therapy for slipped disks “unchanged over time” solved the problem, with disk disruption or a significant reduction in the size of the prolapsed disk material extruded into the spinal canal. PMID:27066816

  14. Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

    Science.gov (United States)

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Chumsae, Chris

    2009-12-01

    Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly.

  15. One size does not fit all: evaluating an intervention to reduce antibiotic prescribing for acute bronchitis.

    Science.gov (United States)

    Ackerman, Sara L; Gonzales, Ralph; Stahl, Melissa S; Metlay, Joshua P

    2013-11-04

    Overuse of antibiotics for upper respiratory tract infections (URIs) and acute bronchitis is a persistent and vexing problem. In the U.S., more than half of all patients with upper respiratory tract infections and acute bronchitis are treated with antibiotics annually, despite the fact that most cases are viral in etiology and are not responsive to antibiotics. Interventions aiming to reduce unnecessary antibiotic prescribing have had mixed results, and successes have been modest. The objective of this evaluation is to use mixed methods to understand why a multi-level intervention to reduce antibiotic prescribing for acute bronchitis among primary care providers resulted in measurable improvement in only one third of participating clinicians. Clinician perspectives on print-based and electronic intervention strategies, and antibiotic prescribing more generally, were elicited through structured telephone surveys at high and low performing sites after the first year of intervention at the Geisinger Health System in Pennsylvania (n = 29). Compared with a survey on antibiotic use conducted 10 years earlier, clinicians demonstrated greater awareness of antibiotic resistance and how it is impacted by individual prescribing decisions-including their own. However, persistent perceived barriers to reducing prescribing included patient expectations, time pressure, and diagnostic uncertainty, and these factors were reported as differentially undermining specific intervention components' effectiveness. An exam room poster depicting a diagnostic algorithm was the most popular strategy. Future efforts to reduce antibiotic prescribing should address multi-level barriers identified by clinicians and tailor strategies to differences at individual clinician and group practice levels, focusing in particular on changing how patients and providers make decisions together about antibiotic use.

  16. Face inversion and acquired prosopagnosia reduce the size of the perceptual field of view.

    Science.gov (United States)

    Van Belle, Goedele; Lefèvre, Philippe; Rossion, Bruno

    2015-03-01

    Using a gaze-contingent morphing approach, we asked human observers to choose one of two faces that best matched the identity of a target face: one face corresponded to the reference face's fixated part only (e.g., one eye), the other corresponded to the unfixated area of the reference face. The face corresponding to the fixated part was selected significantly more frequently in the inverted than in the upright orientation. This observation provides evidence that face inversion reduces an observer's perceptual field of view, even when both upright and inverted faces are displayed at full view and there is no performance difference between these conditions. It rules out an account of the drop of performance for inverted faces--one of the most robust effects in experimental psychology--in terms of a mere difference in local processing efficiency. A brain-damaged patient with pure prosopagnosia, viewing only upright faces, systematically selected the face corresponding to the fixated part, as if her perceptual field was reduced relative to normal observers. Altogether, these observations indicate that the absence of visual knowledge reduces the perceptual field of view, supporting an indirect view of visual perception. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A reduced estimate of the number of kilometre-sized near-Earth asteroids.

    Science.gov (United States)

    Rabinowitz, D; Helin, E; Lawrence, K; Pravdo, S

    2000-01-13

    Near-Earth asteroids are small (diameters 1 km has been estimated to be in the range 1,000-2,000, which translates to an approximately 1% chance of a catastrophic collision with the Earth in the next millennium. These numbers are, however, poorly constrained because of the limitations of previous searches using photographic plates. (One kilometre is below the size of a body whose impact on the Earth would produce global effects.) Here we report an analysis of our survey for near-Earth asteroids that uses improved detection technologies. We find that the total number of asteroids with diameters > 1 km is about half the earlier estimates. At the current rate of discovery of near-Earth asteroids, 90% will probably have been detected within the next 20 years.

  18. Reducing Size, Weight, and Power (SWaP) of Perception Systems in Small Autonomous Aerial Systems

    Science.gov (United States)

    Jones, Kennie H.; Gross, Jason

    2014-01-01

    The objectives are to examine recent trends in the reduction of size, weight, and power (SWaP) requirements of sensor systems for environmental perception and to explore new technology that may overcome limitations in current systems. Improving perception systems to facilitate situation awareness is critical in the move to introduce increasing autonomy in aerial systems. Whether the autonomy is in the current state-of-the-art of increasing automation or is enabling cognitive decisions that facilitate adaptive behavior, collection of environmental information and fusion of that information into knowledge that can direct actuation is imperative to decisions resulting in appropriate behavior. Artificial sensory systems such as cameras, radar, LIDAR, and acoustic sensors have been in use on aircraft for many years but, due to the large size and weight of the airplane and electrical power made available through powerful engines, the SWaP requirements of these sensors was inconsequential. With the proliferation of Remote Piloted Vehicles (RPV), the trend is in significant reduction in SWaP of the vehicles. This requires at least an equivalent reduction in SWaP for the sensory systems. A survey of some currently available sensor systems and changing technology will reveal the trend toward reduction of SWaP of these systems and will predict future reductions. A new technology will be introduced that provides an example of a desirable new trend. A new device replaces multiple conventional sensory devices facilitating synchronization, localization, altimetry, collision avoidance, terrain mapping, and data communication in a single integrated, small form-factor, extremely lightweight, and low power device that it is practical for integration into small autonomous vehicles and can facilitate cooperative behavior. The technology is based on Ultra WideBand (UWB) radio using short pulses of energy rather than continuous sine waves. The characteristics of UWB yield several

  19. Individual thorax geometry reduces position and size differences in reconstructed images of electrical impedance tomography.

    Science.gov (United States)

    Zhao, Zhanqi; Frerichs, Inéz; Pulletz, Sven; Müller-Lisse, Ullrich; Möller, Knut

    2014-01-01

    Due to the ill-posed problem, the electrical impedance within the thorax cannot be exactly reconstructed. The aim of our study was to prove that reconstruction with individual thorax geometry improved the quality of EIT (electrical impedance tomography) images. Seven mechanically ventilated patients with acute respiratory distress syndrome were examined by EIT. The thorax contours were determined from routine computed tomography (CT) images based on automatic threshold filtering. EIT raw data was reconstructed offline with (1) back-projection with circular forward model; (2) GREIT reconstruction method with circular forward model and (3) GREIT with individual thorax geometry. The resulting EIT images were compared to rescaled CT images. The distance between the lung contour and the thorax contour was calculated for each method and the differences to that in CT were denoted as position differences. Shape differences was defined as the ratio of thorax (or lungs) size in EIT and that in rescaled CT. Method (3) has the smallest position differences (6.6 ± 2.8, 5.3 ± 3.3, 2.3 ± 1.4 in pixel, for each reconstruction method respectively; mean ± SD). The thorax and lungs sizes in the transformed CT images were 514 ± 73 and 177 ± 39. Shape differences of thorax were 1.81 ± 0.26, 1.81 ± 0.26, 1.10 ± 0.12 and that of lungs were 1.69 ± 0.45, 1.52 ± 0.45, 1.34 ± 0.35 for each method respectively. The reconstructed images using the GREIT method with individual thorax geometry were more realistic. Improvement of EIT image quality may foster the acceptance of EIT in routine clinical use.

  20. Extracellular Vesicles in Cardiovascular Theranostics

    OpenAIRE

    Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S.; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro,Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells pla...

  1. The Harmonic Minor Scale Provides an Optimum Way of Reducing Average Melodic Interval Size, Consistent with Sad Affect Cues

    Directory of Open Access Journals (Sweden)

    David Huron

    2013-08-01

    Full Text Available Small pitch movement is known to characterize sadness in speech prosody. Small melodic interval sizes have also been observed in nominally sad music—at least in the case of Western music. Starting with melodies in the major mode, a study is reported which examines the effect of different scale modifications on the average interval size. Compared with all other possible scale modifications, lowering the third and sixth scale tones from the major scale is shown to provide an optimum or near optimum way of reducing the average melodic interval size for a large diverse sample of major-mode melodies. The results are consistent with the view that Western melodic organization and the major-minor polarity are co-adapted, and that the structure of the minor mode contributes to the evoking, expressing or representation of sadness for listeners enculturated to the major scale.

  2. RNA in extracellular vesicles.

    Science.gov (United States)

    Kim, Kyoung Mi; Abdelmohsen, Kotb; Mustapic, Maja; Kapogiannis, Dimitrios; Gorospe, Myriam

    2017-07-01

    Cells release a range of membrane-enclosed extracellular vesicles (EVs) into the environment. Among them, exosomes and microvesicles (collectively measuring 40-1000 nm in diameter) carry proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been proposed to serve as intercellular mediators of communication. EVs transport cellular materials in many physiologic processes, including differentiation, stem cell homeostasis, immune responses, and neuronal signaling. EVs are also increasingly recognized as having a direct role in pathologies such as cancer and neurodegeneration. Accordingly, EVs have been the focus of intense investigation as biomarkers of disease, prognostic indicators, and even therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs (messenger RNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The rising attention to EV-resident RNAs as biomarkers stems from the fact that RNAs can be detected at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we discuss EV RNAs in cancer and neurodegeneration, two major age-associated disease processes. WIREs RNA 2017, 8:e1413. doi: 10.1002/wrna.1413 For further resources related to this article, please visit the WIREs website. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  3. Extracellular Vesicles in Renal Pathophysiology.

    Science.gov (United States)

    Pomatto, Margherita A C; Gai, Chiara; Bussolati, Benedetta; Camussi, Giovanni

    2017-01-01

    Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

  4. Extracellular vesicles in renal disease.

    Science.gov (United States)

    Karpman, Diana; Ståhl, Anne-Lie; Arvidsson, Ida

    2017-09-01

    Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.

  5. Intermittent exposure to reduced oxygen levels affects prey size selection and consumption in swimming crab Thalamita danae Stimpson.

    Science.gov (United States)

    Shin, P K S; Cheung, P H; Yang, F Y; Cheung, S G

    2005-01-01

    Portunid crabs Thalamita danae (carapace width: 46-56 mm) were exposed to low oxygen level (4.0 mg O2 l(-1)) and hypoxia (1.5 mg O2 l(-1)) for 6 h each day with three size classes (large: 15.0-19.9 mm, medium: 10.0-14.9 mm, small: 5.0-9.9 mm) of mussels Brachidontes variabilis offered as food. Consumption rate, prey size preference, and prey handling including breaking time, handling time, eating time and prey value, were studied during the time the crabs were exposed to reduced oxygen levels and results were compared with the crabs maintained at high oxygen level (8.0 mg O2 l(-1)) throughout the experiment. Consumption of mussels from all size classes was significantly higher at high oxygen level than at reduced oxygen levels. No mussel size preference was observed for crabs exposed to 4.0 or 8.0 mg O2 l(-1) but those crabs exposed to 1.5 mg O2 l(-1) preferred medium mussels. Both breaking time and handling time increased with mussel size but did not vary with oxygen level. Prey value of each mussel consumed (mg dry wt eaten crab(-1) s(-1)) was calculated by dividing the estimated dry weight of the mussel by the observed handling time. Mean prey value varied significantly with mussel size, with values obtained for large mussels being higher than small mussels at 4.0 and 8.0 mg O2 l(-1); the effect of oxygen level, however, was insignificant. In view of portunid crabs as major predators of mussels, results may help explain dominance of mussels in eutrophic harbours in Hong Kong.

  6. Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality.

    Science.gov (United States)

    Su, Wan-Chih; Gettel, Douglas L; Chabanon, Morgan; Rangamani, Padmini; Parikh, Atul N

    2018-01-17

    The ability of large macromolecules to exhibit nontrivial deviations in colligative properties of their aqueous solutions is well-appreciated in polymer physics. Here, we show that this colligative nonideality subjects giant lipid vesicles containing inert macromolecular crowding agents to osmotic pressure differentials when bathed in small-molecule osmolytes at comparable concentrations. The ensuing influx of water across the semipermeable membrane induces characteristic swell-burst cycles: here, cyclical and damped oscillations in size, tension, and membrane phase separation occur en route to equilibration. Mediated by synchronized formation of transient pores, these cycles orchestrate pulsewise ejection of macromolecules from the vesicular interior reducing the osmotic differential in a stepwise manner. These experimental findings are fully corroborated by a theoretical model derived by explicitly incorporating the contributions of the solution viscosity, solute diffusivity, and the colligative nonideality of the osmotic pressure in a previously reported continuum description. Simulations based on this model account for the differences in the details of the noncolligatively induced swell-burst cycles, including numbers and periods of the repeating cycles, as well as pore lifetimes. Taken together, our observations recapitulate behaviors of vesicles and red blood cells experiencing sudden osmotic shocks due to large (hundreds of osmolars) differences in the concentrations of small molecule osmolytes and link intravesicular macromolecular crowding with membrane remodeling. They further suggest that any tendency for spontaneous overcrowding in single giant vesicles is opposed by osmotic stresses and requires independent specific interactions, such as associative chemical interactions or those between the crowders and the membrane boundary.

  7. Comparing two psychological interventions in reducing impulsive processes of eating behaviour: effects on self-selected portion size.

    Science.gov (United States)

    van Koningsbruggen, Guido M; Veling, Harm; Stroebe, Wolfgang; Aarts, Henk

    2014-11-01

    Palatable food, such as sweets, contains properties that automatically trigger the impulse to consume it even when people have goals or intentions to refrain from consuming such food. We compared the effectiveness of two interventions in reducing the portion size of palatable food that people select for themselves. Specifically, the use of dieting implementation intentions that reduce behaviour towards palatable food via top-down implementation of a dieting goal was pitted against a stop-signal training that changes the impulse-evoking quality of palatable food from bottom-up. We compared the two interventions using a 2 × 2 factorial design. Participants completed a stop-signal training in which they learned to withhold a behavioural response upon presentation of tempting sweets (vs. control condition) and formed implementation intentions to diet (vs. control condition). Selected portion size was measured in a sweet-shop-like environment (Experiment 1) and through a computerized snack dispenser (Experiment 2). Both interventions reduced the amount of sweets selected in the sweet shop environment (Experiment 1) and the snack dispenser (Experiment 2). On average, participants receiving an intervention selected 36% (Experiment 1) and 51% (Experiment 2) fewer sweets than control participants. In both studies, combining the interventions did not lead to additive effects: Employing one of the interventions appears to successfully eliminate instrumental behaviour towards tempting food, making the other intervention redundant. Both interventions reduce self-selected portion size, which is considered a major contributor to the current obesity epidemic. What is already known on this subject? Exposure to temptations, such as unhealthy palatable food, often frustrates people's attainment of long-term health goals. Current approaches to self-control suggest that this is partly because temptations automatically trigger impulsive or hedonic processes that override the

  8. Endoluminal gingival fibroblast transfer reduces the size of rabbit carotid aneurisms via elastin repair.

    Science.gov (United States)

    Durand, Eric; Fournier, Benjamin; Couty, Ludovic; Lemitre, Mathilde; Achouh, Paul; Julia, Pierre; Trinquart, Ludovic; Fabiani, Jean Noel; Seguier, Sylvie; Gogly, Bruno; Coulomb, Bernard; Lafont, Antoine

    2012-08-01

    Matrix metalloproteinase-9 is considered to play a pivotal role in aneurismal formation. We showed that gingival fibroblasts (GF) in vitro reduced matrix metalloproteinase-9 activity via increased secretion of tissue inhibitor of metalloproteinase 1. We aimed to evaluate in vivo the efficacy of GF transplantation to reduce aneurism development in a rabbit model. Seventy rabbit carotid aneurisms were induced by elastase infusion. Four weeks later, GF, dermal fibroblast, or culture medium (DMEM) were infused into established aneurisms. Viable GF were abundantly detected in the transplanted arteries 3 months after seeding. GF engraftment resulted in a significant reduction of carotid aneurisms (decrease of 23.3% [P<0.001] and 17.6% [P=0.01] of vessel diameter in GF-treated arteries, 1 and 3 months after cell therapy, respectively), whereas vessel diameter of control DMEM and dermal fibroblast-treated arteries increased. GF inhibited matrix metalloproteinase-9 activity by tissue inhibitor of metalloproteinase 1 overexpression and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase 1 complex formation, induced elastin repair, and increased elastin density in the media compared with DMEM-treated arteries (38.2 versus 18.0%; P=0.02). Elastin network GF-induced repair was inhibited by tissue inhibitor of metalloproteinase 1 blocking peptide. Our results demonstrate that GF transplantation results in significant aneurism reduction and elastin repair. This strategy may be attractive because GF are accessible and remain viable within the grafted tissue.

  9. Overexpression of the Arabidopsis gai gene in apple significantly reduces plant size.

    Science.gov (United States)

    Zhu, L H; Li, X Y; Welander, M

    2008-02-01

    Genetic engineering is an attractive method to obtain dwarf plants in order to eliminate the extensive use of growth retardants in horticultural crop production. In this study, we evaluated the potential of using the Arabidopsis gai (gibberellic acid insensitive) gene to dwarf apple trees. The gai gene under 35S promoter was introduced in the apple rootstock A2 and the cultivars Gravenstein and McIntosh through Agrobacterium-mediated transformation. One transgenic clone was recovered for Gravenstein and McIntosh, and several transgenic clones for A2, confirmed by Southern blot analysis. Two weak bands were detected by Southern blot analysis in all the untransformed controls, possibly indicating the existence of the internal GAI gene in apple. Most of the transgenic plants showed reduced growth in vitro. Growth analyses in the greenhouse showed a clear reduction in stem length, internode length and node number for the dwarf clones. The normal phenotype of some transgenic clones appears to be associated with silencing of the introduced gai gene, confirmed by RT-PCR analysis. In general, transgenic clones showed reduced rooting ability, especially for the extremely compact ones.

  10. Efficacy of a 2% climbazole shampoo for reducing Malassezia population sizes on the skin of naturally infected dogs.

    Science.gov (United States)

    Cavana, P; Petit, J-Y; Perrot, S; Guechi, R; Marignac, G; Reynaud, K; Guillot, J

    2015-12-01

    Shampoo therapy is often recommended for the control of Malassezia overgrowth in dogs. The aim of this study was to evaluate the in vivo activity of a 2% climbazole shampoo against Malassezia pachydermatis yeasts in naturally infected dogs. Eleven research colony Beagles were used. The dogs were distributed randomly into two groups: group A (n=6) and group B (n=5). Group A dogs were washed with a 2% climbazole shampoo, while group B dogs were treated with a physiological shampoo base. The shampoos were applied once weekly for two weeks. The population size of Malassezia yeasts on skin was determined by fungal culture through modified Dixon's medium contact plates pressed on left concave pinna, axillae, groins, perianal area before and after shampoo application. Samples collected were compared by Wilcoxon rank sum test. Samples collected after 2% climbazole shampoo application showed a significant and rapid reduction of Malassezia population sizes. One hour after the first climbazole shampoo application, Malassezia reduction was already statistically significant and 15 days after the second climbazole shampoo, Malassezia population sizes were still significantly decreased. No significant reduction of Malassezia population sizes was observed in group B dogs. The application of a 2% climbazole shampoo significantly reduced Malassezia population sizes on the skin of naturally infected dogs. Application of 2% climbazole shampoo may be useful for the control of Malassezia overgrowth and it may be also proposed as prevention when recurrences are frequent. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Body size and human energy requirements: reduced mass-specific resting energy expenditure in tall adults.

    Science.gov (United States)

    Heymsfield, Steven B; Childers, Douglas; Beetsch, Joel; Allison, David B; Pietrobelli, Angelo

    2007-11-01

    Two observations favor the presence of a lower mass-specific resting energy expenditure (REE/weight) in taller adult humans: an earlier report of height (H)-related differences in relative body composition; and a combined model based on Quetelet and Kleiber's classic equations suggesting that REE/weight proportional, variantH(-0.5). This study tested the hypothesis stating that mass-specific REE scales negatively to height with a secondary aim exploration of related associations between height, weight (W), surface area (SA), and REE. Two independent data sets (n = 344 and 884) were evaluated, both with REE measured by indirect calorimetry and the smaller of the two including fat estimates by dual-energy X-ray absorptiometry. Results support Quetelet's equation (W proportional, variantH(2)), but Kleiber's equation approached the interspecific mammal form (REE proportional, variantW(0.75)) only after adding adiposity measures to weight and age as REE predictors. REE/weight scaled as H( approximately (-0.5)) in support of the hypothesis with P values ranging from 0.17 to <0.001. REE and SA both scaled as H( approximately 1.5), and REE/SA was nonsignificantly correlated with height in all groups. These observations suggest that adiposity needs to be considered when evaluating the intraspecific scaling of REE to weight; that relative to their weight, taller subjects require a lower energy intake for replacing resting heat losses than shorter subjects; that fasting endurance, approximated as fat mass/REE, increases as H(0.5); and that thermal balance is maintained independent of stature by evident stable associations between resting heat production and capacity of external heat release. These observations have implications for the modeling of adult human energy requirements and associate with anthropological concepts founded on body size.

  12. Effective size of a wild salmonid population is greatly reduced by hatchery supplementation.

    Science.gov (United States)

    Christie, M R; Marine, M L; French, R A; Waples, R S; Blouin, M S

    2012-10-01

    Many declining and commercially important populations are supplemented with captive-born individuals that are intentionally released into the wild. These supplementation programs often create large numbers of offspring from relatively few breeding adults, which can have substantial population-level effects. We examined the genetic effects of supplementation on a wild population of steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, by matching 12 run-years of hatchery steelhead back to their broodstock parents. We show that the effective number of breeders producing the hatchery fish (broodstock parents; N(b)) was quite small (harmonic mean N(b)=25 fish per brood-year vs 373 for wild fish), and was exacerbated by a high variance in broodstock reproductive success among individuals within years. The low N(b) caused hatchery fish to have decreased allelic richness, increased average relatedness, more loci in linkage disequilibrium and substantial levels of genetic drift in comparison with their wild-born counterparts. We also documented a substantial Ryman-Laikre effect whereby the additional hatchery fish doubled the total number of adult fish on the spawning grounds each year, but cut the effective population size of the total population (wild and hatchery fish combined) by nearly two-thirds. We further demonstrate that the Ryman-Laikre effect is most severe in this population when (1) >10% of fish allowed onto spawning grounds are from hatcheries and (2) the hatchery fish have high reproductive success in the wild. These results emphasize the trade-offs that arise when supplementation programs attempt to balance disparate goals (increasing production while maintaining genetic diversity and fitness).

  13. Growth regulators in reducing the size of orchid Fire-of-Star for commercialization in vase

    Directory of Open Access Journals (Sweden)

    Patricia Reiners Carvalho

    2016-05-01

    Full Text Available Fire-of-star (Epidendrum radicans Pav. ex Lindl. is a terrestrial orchid, native to Brazil, tussocks with leafy stems, always with many adventitious roots, releasing its long inflorescence with about 1.0 m from the apex of the stem, showing great potential in floriculture, but long flowering stem complicates their marketing vase. The objective of this study was to evaluate the effect of paclobutrazol (PBZ and mepiquat chloride (CLM the reduction of the size of the orchid E. radicans. Plants with an average height of 15 cm were cultivated in a greenhouse with 50% shading. The growth regulators used were PBZ at doses of 0; 5; 10; 15 and 20 mg L-1, and the CLM at doses of 0; 1; 2; 3; 4 and 5 mg L-1. The frequency of application was fortnightly, totaling ten applications. The experiment was installed on a randomized complete blocks, one block to the PBZ with 5 treatments and 10 replications and another block to the CLM, with 6 treatments and 10 replications. Data were submitted to analysis of variance at 5% probability and significance when seen performed regression analysis. The variables evaluated were number shoots, plant height (cm, number of flower stems and leaf area. The results indicated that E. radicans treated with 5 mg L-1 PBZ were 50% lower in height than the control plants. When CLM treated with a dose of 1 mg L-1 plants were 25% lower in height than the control plants, maintaining its aesthetic characteristics suitable for marketing in vases. Growth regulators in the applied doses did not affect the number of shoots and flower stems. PBZ treated plants had 50% of their leaf area compared to control while those treated with CLM doses remained with the same average leaf area of control.

  14. Soluble epoxide hydrolase gene deletion improves blood flow and reduces infarct size after cerebral ischemia in reproductively senescent female mice

    Directory of Open Access Journals (Sweden)

    Kristen L Zuloaga

    2015-01-01

    Full Text Available Soluble epoxide hydrolase (sEH, a key enzyme in the metabolism of vasodilatory epoxyeicosatrienoic acids (EETs, is sexually dimorphic, suppressed by estrogen, and contributes to underlying sex differences in cerebral blood flow and injury after cerebral ischemia. We tested the hypothesis that sEH inhibition or gene deletion in reproductively senescent (RS female mice would increase cerebral perfusion and decrease infarct size following stroke. RS (15-18 month old and young (3-4 month old female sEH knockout (sEHKO mice and wild type (WT mice were subjected to 45 min middle cerebral artery occlusion (MCAO with laser Doppler perfusion monitoring. WT mice were treated with vehicle or a sEH inhibitor t-AUCB at the time of reperfusion and every 24hrs thereafter for 3 days. Differences in regional cerebral blood flow were measured in vivo using optical microangiography. Infarct size was measured 3 days after reperfusion. Infarct size and cerebral perfusion 24h after MCAO were not altered by age. Both sEH gene deletion and sEH inhibition increased cortical perfusion 24h after MCAO. Neither sEH gene deletion nor sEH inhibition reduced infarct size in young mice. However, sEH gene deletion, but not sEH inhibition of the hydrolase domain of the enzyme, decreased infarct size in RS mice. Results of these studies show that sEH gene deletion and sEH inhibition enhance cortical perfusion following MCAO and sEH gene deletion reduces damage after ischemia in RS female mice; however this neuroprotection in absent is young mice.

  15. Acute administration of cannabidiol in vivo suppresses ischaemia-induced cardiac arrhythmias and reduces infarct size when given at reperfusion

    Science.gov (United States)

    Walsh, Sarah K; Hepburn, Claire Y; Kane, Kathleen A; Wainwright, Cherry L

    2010-01-01

    Background and purpose: Cannabidiol (CBD) is a phytocannabinoid, with anti-apoptotic, anti-inflammatory and antioxidant effects and has recently been shown to exert a tissue sparing effect during chronic myocardial ischaemia and reperfusion (I/R). However, it is not known whether CBD is cardioprotective in the acute phase of I/R injury and the present studies tested this hypothesis. Experimental approach: Male Sprague-Dawley rats received either vehicle or CBD (10 or 50 µg·kg−1 i.v.) 10 min before 30 min coronary artery occlusion or CBD (50 µg·kg−1 i.v.) 10 min before reperfusion (2 h). The appearance of ventricular arrhythmias during the ischaemic and immediate post-reperfusion periods were recorded and the hearts excised for infarct size determination and assessment of mast cell degranulation. Arterial blood was withdrawn at the end of the reperfusion period to assess platelet aggregation in response to collagen. Key results: CBD reduced both the total number of ischaemia-induced arrhythmias and infarct size when administered prior to ischaemia, an effect that was dose-dependent. Infarct size was also reduced when CBD was given prior to reperfusion. CBD (50 µg·kg−1 i.v.) given prior to ischaemia, but not at reperfusion, attenuated collagen-induced platelet aggregation compared with control, but had no effect on ischaemia-induced mast cell degranulation. Conclusions and implications: This study demonstrates that CBD is cardioprotective in the acute phase of I/R by both reducing ventricular arrhythmias and attenuating infarct size. The anti-arrhythmic effect, but not the tissue sparing effect, may be mediated through an inhibitory effect on platelet activation. PMID:20590615

  16. Salt, shake, fuse--giant hybrid polymer/lipid vesicles through mechanically activated fusion.

    Science.gov (United States)

    Henderson, Ian M; Paxton, Walter F

    2014-03-24

    Large (200 nm) poly(ethylene oxide)-b-poly(butadiene) polymer vesicles fuse into giant (>1 μm) vesicles with mild agitation in dilute aqueous NaCl solutions. This unusual effect is attributed to the salt-induced contraction of the poly(ethylene oxide) corona, reducing steric resistance between vesicles and, with agitation, increasing the probability of contact between the hydrophobic cores of adjacent membranes. In addition, NaCl and agitation facilitated the creation of giant hybrid vesicles from much smaller homogeneous polymersomes and liposomes. Whereas lipid vesicles do not readily fuse with each other under the same circumstances, they did fuse with polymersomes to produce hybrid polymer/lipid vesicles. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Stable, biocompatible lipid vesicle generation by solvent extraction-based droplet microfluidics

    Science.gov (United States)

    Teh, Shia-Yen; Khnouf, Ruba; Fan, Hugh; Lee, Abraham P.

    2011-01-01

    In this paper, we present a microfluidic platform for the continuous generation of stable, monodisperse lipid vesicles 20–110 μm in diameter. Our approach utilizes a microfluidic flow-focusing droplet generation design to control the vesicle size by altering the system’s fluid flow rates to generate vesicles with narrow size distribution. Double emulsions are first produced in consecutive flow-focusing channel geometries and lipid membranes are then formed through a controlled solvent extraction process. Since no strong solvents are used in the process, our method allows for the safe encapsulation and manipulation of an assortment of biological entities, including cells, proteins, and nucleic acids. The vesicles generated by this method are stable and have a shelf life of at least 3 months. Here, we demonstrate the cell-free in vitro synthesis of proteins within lipid vesicles as an initial step towards the development of an artificial cell. PMID:22685501

  18. Reduced size of the amygdala in individuals with 47,XXY and 47,XXX karyotypes.

    Science.gov (United States)

    Patwardhan, Anil J; Brown, Wendy E; Bender, Bruce G; Linden, Mary G; Eliez, Stephan; Reiss, Allan L

    2002-01-08

    The excess of 47,XXX and 47,XXY karyotypes found in cytogenetic screening studies of individuals with schizophrenia has given support for an increased risk of psychiatric illness among men and women with sex chromosomal aneuploidy (SCA). Mesial temporal lobe structures, including the amygdala and hippocampus, are thought to be associated with abnormalities of mood and behavior in humans and in the neurobiology of schizophrenia. This study focuses on variations in volumes of mesial temporal lobe structures in men and women with SCA. Utilizing an unselected birth cohort of subjects with SCA and high-resolution magnetic resonance imaging (MRI), we investigated the neuroanatomical consequences of a supernumerary X chromosome on the morphology of the amygdala and hippocampus. Regional and total brain volumes were measured in 10 subjects with 47,XXY, 10 subjects with 47,XXX, and 20 euploid controls. Amygdala volumes were significantly reduced in men with 47,XXY, compared to control men, while the decrease in women with 47,XXX was not as pronounced. Hippocampus volumes were preserved in both groups, compared to same-gender controls. Longitudinal studies of SCA individuals have shown an increased incidence of mild psychopathology and behavioral dysfunction in men with 47,XXY and more overt psychiatric illness in women with 47,XXX, compared to control populations. The alteration in amygdala volumes in individuals with a supernumerary X chromosome may provide a neuroanatomic basis for these findings. Copyright 2001 Wiley-Liss, Inc.

  19. Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools.

    Science.gov (United States)

    Geng, Junhua; Wang, Liping; Lee, Joo Yeun; Chen, Chun-Kan; Chang, Karen T

    2016-08-24

    The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for Synaptojanin in

  20. Reduced population size does not affect the mating strategy of a vulnerable and endemic seabird

    Science.gov (United States)

    Nava, Cristina; Neves, Verónica C.; Andris, Malvina; Dubois, Marie-Pierre; Jarne, Philippe; Bolton, Mark; Bried, Joël

    2017-12-01

    Bottleneck episodes may occur in small and isolated animal populations, which may result in decreased genetic diversity and increased inbreeding, but also in mating strategy adjustment. This was evaluated in the vulnerable and socially monogamous Monteiro's Storm-petrel Hydrobates monteiroi, a seabird endemic to the Azores archipelago which has suffered a dramatic population decline since the XVth century. To do this, we conducted a genetic study (18 microsatellite markers) in the population from Praia islet, which has been monitored over 16 years. We found no evidence that a genetic bottleneck was associated with this demographic decline. Monteiro's Storm-petrels paired randomly with respect to genetic relatedness and body measurements. Pair fecundity was unrelated to genetic relatedness between partners. We detected only two cases of extra-pair parentage associated with an extra-pair copulation (out of 71 offspring). Unsuccessful pairs were most likely to divorce the next year, but genetic relatedness between pair mates and pair breeding experience did not influence divorce. Divorce enabled individuals to improve their reproductive performances after re-mating only when the new partner was experienced. Re-pairing with an experienced partner occurred more frequently when divorcees changed nest than when they retained their nest. This study shows that even in strongly reduced populations, genetic diversity can be maintained, inbreeding does not necessarily occur, and random pairing is not risky in terms of pair lifetime reproductive success. Given, however, that we found no clear phenotypic mate choice criteria, the part played by non-morphological traits should be assessed more accurately in order to better understand seabird mating strategies.

  1. Cinacalcet effectively reduces parathyroid hormone secretion and gland volume regardless of pretreatment gland size in patients with secondary hyperparathyroidism.

    Science.gov (United States)

    Komaba, Hirotaka; Nakanishi, Shohei; Fujimori, Akira; Tanaka, Motoko; Shin, Jeongsoo; Shibuya, Koji; Nishioka, Masato; Hasegawa, Hirohito; Kurosawa, Takeshi; Fukagawa, Masafumi

    2010-12-01

    Cinacalcet is effective in reducing serum parathyroid hormone (PTH) in patients with secondary hyperparathyroidism. However, it has not been proven whether parathyroid gland size predicts response to therapy and whether cinacalcet is capable of inducing a reduction in parathyroid volume. This 52-week, multicenter, open-label study enrolled hemodialysis patients with moderate to severe secondary hyperparathyroidism (intact PTH >300 pg/ml). Doses of cinacalcet were adjusted between 25 and 100 mg to achieve intact PTH size at baseline, week 26, and week 52. Findings were also compared with those of historical controls. Of the 81 subjects enrolled, 56 had parathyroid glands smaller than 500 mm(3) (group S) and 25 had at least one enlarged gland larger than 500 mm(3) (group L). Treatment with cinacalcet effectively decreased intact PTH by 55% from baseline in group S and by 58% in group L. A slightly greater proportion of patients in group S versus group L achieved an intact PTH 30% reduction from baseline (88 versus 78%), but this was not statistically significant. Cinacalcet therapy also resulted in a significant reduction in parathyroid gland volume regardless of pretreatment size, which was in sharp contrast to historical controls (n = 87) where parathyroid gland volume progressively increased with traditional therapy alone. Cinacalcet effectively decreases serum PTH levels and concomitantly reduces parathyroid gland volume, even in patients with marked parathyroid hyperplasia.

  2. Hypothyroidism Reduces the Size of Ovarian Follicles and Promotes Hypertrophy of Periovarian Fat with Infiltration of Macrophages in Adult Rabbits.

    Science.gov (United States)

    Rodríguez-Castelán, J; Méndez-Tepepa, M; Carrillo-Portillo, Y; Anaya-Hernández, A; Rodríguez-Antolín, J; Zambrano, E; Castelán, F; Cuevas-Romero, E

    2017-01-01

    Ovarian failure is related to dyslipidemias and inflammation, as well as to hypertrophy and dysfunction of the visceral adipose tissue (VAT). Although hypothyroidism has been associated with obesity, dyslipidemias, and inflammation in humans and animals, its influence on the characteristics of ovarian follicles in adulthood is scarcely known. Control and hypothyroid rabbits were used to analyze the ovarian follicles, expression of aromatase in the ovary, serum concentration of lipids, leptin, and uric acid, size of adipocytes, and infiltration of macrophages in the periovarian VAT. Hypothyroidism did not affect the percentage of functional or atretic follicles. However, it reduced the size of primary, secondary, and tertiary follicles considered as large and the expression of aromatase in the ovary. This effect was associated with high serum concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C). In addition, hypothyroidism induced hypertrophy of adipocytes and a major infiltration of CD68+ macrophages into the periovarian VAT. Our results suggest that the reduced size of ovarian follicles promoted by hypothyroidism could be associated with dyslipidemias, hypertrophy, and inflammation of the periovarian VAT. Present findings may be useful to understand the influence of hypothyroidism in the ovary function in adulthood.

  3. Hypothyroidism Reduces the Size of Ovarian Follicles and Promotes Hypertrophy of Periovarian Fat with Infiltration of Macrophages in Adult Rabbits

    Directory of Open Access Journals (Sweden)

    J. Rodríguez-Castelán

    2017-01-01

    Full Text Available Ovarian failure is related to dyslipidemias and inflammation, as well as to hypertrophy and dysfunction of the visceral adipose tissue (VAT. Although hypothyroidism has been associated with obesity, dyslipidemias, and inflammation in humans and animals, its influence on the characteristics of ovarian follicles in adulthood is scarcely known. Control and hypothyroid rabbits were used to analyze the ovarian follicles, expression of aromatase in the ovary, serum concentration of lipids, leptin, and uric acid, size of adipocytes, and infiltration of macrophages in the periovarian VAT. Hypothyroidism did not affect the percentage of functional or atretic follicles. However, it reduced the size of primary, secondary, and tertiary follicles considered as large and the expression of aromatase in the ovary. This effect was associated with high serum concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C. In addition, hypothyroidism induced hypertrophy of adipocytes and a major infiltration of CD68+ macrophages into the periovarian VAT. Our results suggest that the reduced size of ovarian follicles promoted by hypothyroidism could be associated with dyslipidemias, hypertrophy, and inflammation of the periovarian VAT. Present findings may be useful to understand the influence of hypothyroidism in the ovary function in adulthood.

  4. Hypothyroidism Reduces the Size of Ovarian Follicles and Promotes Hypertrophy of Periovarian Fat with Infiltration of Macrophages in Adult Rabbits

    Science.gov (United States)

    Rodríguez-Castelán, J.; Méndez-Tepepa, M.; Carrillo-Portillo, Y.; Anaya-Hernández, A.; Zambrano, E.

    2017-01-01

    Ovarian failure is related to dyslipidemias and inflammation, as well as to hypertrophy and dysfunction of the visceral adipose tissue (VAT). Although hypothyroidism has been associated with obesity, dyslipidemias, and inflammation in humans and animals, its influence on the characteristics of ovarian follicles in adulthood is scarcely known. Control and hypothyroid rabbits were used to analyze the ovarian follicles, expression of aromatase in the ovary, serum concentration of lipids, leptin, and uric acid, size of adipocytes, and infiltration of macrophages in the periovarian VAT. Hypothyroidism did not affect the percentage of functional or atretic follicles. However, it reduced the size of primary, secondary, and tertiary follicles considered as large and the expression of aromatase in the ovary. This effect was associated with high serum concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C). In addition, hypothyroidism induced hypertrophy of adipocytes and a major infiltration of CD68+ macrophages into the periovarian VAT. Our results suggest that the reduced size of ovarian follicles promoted by hypothyroidism could be associated with dyslipidemias, hypertrophy, and inflammation of the periovarian VAT. Present findings may be useful to understand the influence of hypothyroidism in the ovary function in adulthood. PMID:28133606

  5. Investigation of the InAs/GaAs Quantum Dots’ Size: Dependence on the Strain Reducing Layer’s Position

    Directory of Open Access Journals (Sweden)

    Manel Souaf

    2015-07-01

    Full Text Available This work reports on theoretical and experimental investigation of the impact of InAs quantum dots (QDs position with respect to InGaAs strain reducing layer (SRL. The investigated samples are grown by molecular beam epitaxy and characterized by photoluminescence spectroscopy (PL. The QDs optical transition energies have been calculated by solving the three dimensional Schrödinger equation using the finite element methods and taking into account the strain induced by the lattice mismatch. We have considered a lens shaped InAs QDs in a pure GaAs matrix and either with InGaAs strain reducing cap layer or underlying layer. The correlation between numerical calculation and PL measurements allowed us to track the mean buried QDs size evolution with respect to the surrounding matrix composition. The simulations reveal that the buried QDs’ realistic size is less than that experimentally driven from atomic force microscopy observation. Furthermore, the average size is found to be slightly increased for InGaAs capped QDs and dramatically decreased for QDs with InGaAs under layer.

  6. Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

    Science.gov (United States)

    Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

    2014-10-01

    To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. The Influence of Vesicle Shape and Medium Conductivity on Possible Electrofusion under a Pulsed Electric Field.

    Science.gov (United States)

    Liu, Linying; Mao, Zheng; Zhang, Jianhua; Liu, Na; Liu, Qing Huo

    2016-01-01

    The effects of electric field on lipid membrane and cells have been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest due to their wide use in cell biology and biotechnology. However, numerical studies on the electrofusion of cells (or vesicles) with different deformed shapes are still rare. Vesicle, being of cell size, can be treated as a simple model of cell to investigate the behaviors of cell in electric field. Based on the finite element method, we investigate the effect of vesicle shape on electrofusion of contact vesicles in various medium conditions. The transmembrane voltage (TMV) and pore density induced by a pulsed field are examined to analyze the possibility of vesicle fusion. In two different medium conditions, the prolate shape is observed to have selective electroporation at the contact area of vesicles when the exterior conductivity is smaller than the interior one; selective electroporation is more inclined to be found at the poles of the oblate vesicles when the exterior conductivity is larger than the interior one. Furthermore, we find that when the exterior conductivity is lower than the internal conductivity, the pulse can induce a selective electroporation at the contact area between two vesicles regardless of the vesicle shape. Both of these two findings have important practical applications in guiding electrofusion experiments.

  8. Quantitative analysis of vesicle recycling at the calyx of Held synapse

    Science.gov (United States)

    Qiu, Xufeng; Zhu, Qianwen; Sun, Jianyuan

    2015-01-01

    Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%). PMID:25825725

  9. Extracellular Vesicles in Lung Disease.

    Science.gov (United States)

    Kubo, Hiroshi

    2018-01-01

    Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the pathogenesis of lung diseases. These vesicles include exosomes, ectosomes (ie, microparticles, extracellular vesicles, microvesicles, and shedding vesicles), and apoptotic bodies. Exosomes are generated by inward budding of the membrane (endocytosis), subsequent forming of multivesicular bodies, and release by exocytosis. Ectosomes are formed by outward blebbing from the plasma membrane and are then released by proteolytic cleavage from the cell surface. Apoptotic bodies are generated on apoptotic cell shrinkage and death. Extracellular vesicles are released when the cells are activated or undergo apoptosis under inflammatory conditions. The number and types of released EVs are different according to the pathophysiological status of the disease. Therefore, EVs can be novel biomarkers for various lung diseases. EVs contain several molecules, including proteins, mRNA, microRNA, and DNA; they transfer these molecules to distant recipient cells. Circulating EVs modify the targeted cells and influence the microenvironment of the lungs. For this unique capability, EVs are expected to be a new drug delivery system and a novel therapeutic target. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  10. Remediation of chromium-contaminated water using biogenic nano-sized materials and metal-reducing bacteria.

    Science.gov (United States)

    Seo, Hyunhee; Sun, Eunyoung; Roh, Yul

    2013-06-01

    As an environmental nanotechnology, nano-sized materials have the potential to create novel and effective in-situ and ex-situ treatments for contaminated groundwater due to its high catalytic reactivity, large surface area, and dispersibility. In this study the efficiency of Cr(VI) reduction and immobilization using biotic and abiotic nano-sized materials (NSMs) and metal-reducing bacteria (MRB) was evaluated to remediate Cr(VI)-contaminated groundwater in batch and column tests. The results of this study revealed that the combination of the mixed MRB and bio-FeS/siderite performed the highest efficiency of Cr(VI) reduction and immobilization. Cr(VI) reduction by MRB and NSMs could impact on solubility of Cr(VI) and geochemical changes favorable for precipitation and adsorption.

  11. Use of High-Frequency In-Home Monitoring Data May Reduce Sample Sizes Needed in Clinical Trials.

    Directory of Open Access Journals (Sweden)

    Hiroko H Dodge

    walking speed collected at baseline, 262 subjects are required. Similarly for computer use, 26 subjects are required.Individual-specific thresholds of low functional performance based on high-frequency in-home monitoring data distinguish trajectories of MCI from NC and could substantially reduce sample sizes needed in dementia prevention RCTs.

  12. Methods for the physical characterization and quantification of extracellular vesicles in biological samples.

    Science.gov (United States)

    Rupert, Déborah L M; Claudio, Virginia; Lässer, Cecilia; Bally, Marta

    2017-01-01

    Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed. This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in extracellular vesicle research. The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples. The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Riociguat reduces infarct size and post-infarct heart failure in mouse hearts: insights from MRI/PET imaging.

    Directory of Open Access Journals (Sweden)

    Carmen Methner

    Full Text Available Stimulation of the nitric oxide (NO--soluble guanylate (sGC--protein kinase G (PKG pathway confers protection against acute ischaemia/reperfusion injury, but more chronic effects in reducing post-myocardial infarction (MI heart failure are less defined. The aim of this study was to not only determine whether the sGC stimulator riociguat reduces infarct size but also whether it protects against the development of post-MI heart failure.Mice were subjected to 30 min ischaemia via ligation of the left main coronary artery to induce MI and either placebo or riociguat (1.2 µmol/l were given as a bolus 5 min before and 5 min after onset of reperfusion. After 24 hours, both, late gadolinium-enhanced magnetic resonance imaging (LGE-MRI and (18F-FDG-positron emission tomography (PET were performed to determine infarct size. In the riociguat-treated mice, the resulting infarct size was smaller (8.5 ± 2.5% of total LV mass vs. 21.8% ± 1.7%. in controls, p = 0.005 and LV systolic function analysed by MRI was better preserved (60.1% ± 3.4% of preischaemic vs. 44.2% ± 3.1% in controls, p = 0.005. After 28 days, LV systolic function by echocardiography treated group was still better preserved (63.5% ± 3.2% vs. 48.2% ± 2.2% in control, p = 0.004.Taken together, mice treated acutely at the onset of reperfusion with the sGC stimulator riociguat have smaller infarct size and better long-term preservation of LV systolic function. These findings suggest that sGC stimulation during reperfusion therapy may be a powerful therapeutic treatment strategy for preventing post-MI heart failure.

  14. Extracellular Vesicles in Brain Tumors and Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Federica Ciregia

    2017-08-01

    Full Text Available Extracellular vesicles (EVs can be classified into apoptotic bodies, microvesicles (MVs, and exosomes, based on their origin or size. Exosomes are the smallest and best characterized vesicles which derived from the endosomal system. These vesicles are released from many different cell types including neuronal cells and their functions in the nervous system are investigated. They have been proposed as novel means for intercellular communication, which takes part not only to the normal neuronal physiology but also to the transmission of pathogenic proteins. Indeed, exosomes are fundamental to assemble and transport proteins during development, but they can also transfer neurotoxic misfolded proteins in pathogenesis. The present review will focus on their roles in neurological diseases, specifically brain tumors, such as glioblastoma (GBM, neuroblastoma (NB, medulloblastoma (MB, and metastatic brain tumors and chronic neurodegenerative diseases, such as Alzheimer, Parkinson, multiple sclerosis (MS, amyotrophic lateral sclerosis (ALS, Huntington, and Prion diseseases highlighting their involvement in spreading neurotoxicity, in therapeutics, and in pathogenesis.

  15. Extracellular Vesicles in Brain Tumors and Neurodegenerative Diseases

    Science.gov (United States)

    Ciregia, Federica; Urbani, Andrea; Palmisano, Giuseppe

    2017-01-01

    Extracellular vesicles (EVs) can be classified into apoptotic bodies, microvesicles (MVs), and exosomes, based on their origin or size. Exosomes are the smallest and best characterized vesicles which derived from the endosomal system. These vesicles are released from many different cell types including neuronal cells and their functions in the nervous system are investigated. They have been proposed as novel means for intercellular communication, which takes part not only to the normal neuronal physiology but also to the transmission of pathogenic proteins. Indeed, exosomes are fundamental to assemble and transport proteins during development, but they can also transfer neurotoxic misfolded proteins in pathogenesis. The present review will focus on their roles in neurological diseases, specifically brain tumors, such as glioblastoma (GBM), neuroblastoma (NB), medulloblastoma (MB), and metastatic brain tumors and chronic neurodegenerative diseases, such as Alzheimer, Parkinson, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington, and Prion diseseases highlighting their involvement in spreading neurotoxicity, in therapeutics, and in pathogenesis. PMID:28912682

  16. The quantal secretion of catecholamines is impaired by the accumulation of β-adrenoceptor antagonists into chromaffin cell vesicles

    Science.gov (United States)

    Montesinos, Mónica S; Camacho, Marcial; Machado, J David; Viveros, O Humberto; Beltrán, Beatriz; Borges, Ricardo

    2010-01-01

    Background and purpose: The delayed onset of certain effects of antagonists of β-adrenoceptors (β-blockers), such as lowering arterial blood pressure (several days), cannot be explained solely by their effects on β-adrenoceptors, an action that occurs within minutes. Although several mechanisms have been proposed, none of them explain this temporal delay. This work aimed at providing a new explanation based on the interference of these drugs with the functional accumulation of catecholamines within neurosecretory vesicles. Experimental approach: We used the simultaneous on-line monitoring of catecholamine and labetalol release from bovine isolated chromaffin cells and from rat perfused adrenal glands, as well as single cell amperometry, intracellular electrochemistry, patch amperometry and HPLC. Key results: Using amperometry, three β-blockers, labetalol, atenolol and propranolol, reduced the quantal size of secretory events in chromaffin cells, accompanied by a slowing down of exocytosis. By patch amperometry, we found that treatment with β-blockers also increases the chromaffin vesicle volume, thereby creating a functional dilution of catecholamines. Experiments with intracellular electrochemistry show that vesicles cannot uptake new catecholamines. There was progressive accumulation of labetalol in secretory vesicles of bovine adrenal chromaffin cells, and this β-blocker was co-released with catecholamines from rat and bovine chromaffin tissues. Conclusions and implications: We propose that β-blockers are progressively concentrated into sympathetic secretory vesicles, and interfere with the storage of catecholamines and are co-released with the natural transmitters, resulting in a decrease in the sympathetic tone. This could explain the delayed onset of the hypotensive effects of β-blockers. PMID:20233226

  17. Health at Every Size College Course Reduces Dieting Behaviors and Improves Intuitive Eating, Body Esteem, and Anti-Fat Attitudes.

    Science.gov (United States)

    Humphrey, Lauren; Clifford, Dawn; Neyman Morris, Michelle

    2015-01-01

    To investigate the effects of a Health at Every Size general education course on intuitive eating, body esteem (BES), cognitive behavioral dieting scores, and anti-fat attitudes of college students. Quasi-experimental design with 149 students in intervention (45), comparison (66), or control (46) groups. Analysis of variance and post hoc Tukey adjusted tests were used. Mean scores for total general education course on intuitive eating (P eat (P intuitive eating, body esteem, and anti-fat attitudes and reduced dieting behaviors compared with students in the control and comparison groups. Copyright © 2015 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

  18. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

  19. Cystadenoma of the seminal vesicle

    Directory of Open Access Journals (Sweden)

    Gil Antônio O.

    2003-01-01

    Full Text Available Primary tumors of the seminal vesicle are extremely rare. Among them, there is a spectrum of tumors derived from both epithelium and stroma and so classified as epithelial-stromal tumors. Herein, we report a case of a cystadenoma in a 49-year-old asymptomatic man, detected in a routine ultrasonography for liver disease follow-up. The digital rectal examination detected a large mass anterior to rectum and posterior to bladder. Computed tomography scan and magnetic resonance imaging showed a normal prostate and a 9.0 cm cystic tumor, replacing the left seminal vesicle. The gross appearance and microscopic aspect was compatible with cystadenoma of seminal vesicle. Patient's postoperative recovery was uneventful. He is currently alive, 3 years after the diagnosis, with no signs of recurrence.

  20. When to biopsy seminal vesicles.

    Science.gov (United States)

    Panach-Navarrete, J; García-Morata, F; Hernández-Medina, J A; Martínez-Jabaloyas, J M

    2015-05-01

    The involvement of seminal vesicles in prostate cancer can affect the prognosis and determine the treatment. The objective of this study was to determine whether we could predict its infiltration at the time of the prostate biopsy to know when to indicate the biopsy of the seminal vesicles. observational retrospective study of 466 patients who underwent seminal vesicle biopsy. The indication for this biopsy was a prostate-specific antigen (PSA) level greater than 10 ng/ml or an asymmetric or obliterated prostatoseminal angle. The following variables were included in the analysis: PSA level, PSA density, prostate volume, number of cores biopsied, suspicious rectal examination, and preservation of the prostatoseminal angle, studying its relationship with the involvement of the seminal vesicles. Forty-one patients (8.8%) had infiltrated seminal vesicles and 425 (91.2%) had no involvement. In the univariate analysis, the cases with infiltration had a higher mean PSA level (P 19.60 ng/dL (P < .01) and 2.95 times higher if there is a suspicious rectal examination (P = .014). Furthermore, this probability increases by 1.04 times for each unit of prostate volume lower (P < .01). The ROC curves showed maximum sensitivity and specificity at 19.6 ng/mL for PSA and 0.39 for PSA density. In this series, greater involvement of seminal vesicles was associated with a PSA level ≥20 ng/ml, a suspicious rectal examination and a lack of prostatoseminal angle preservation. Copyright © 2014 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  1. Cryo-electron microscopy of extracellular vesicles in fresh plasma.

    Science.gov (United States)

    Yuana, Yuana; Koning, Roman I; Kuil, Maxim E; Rensen, Patrick C N; Koster, Abraham J; Bertina, Rogier M; Osanto, Susanne

    2013-12-31

    Extracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited. To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM). Fresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM. EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25-260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules. Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

  2. Cryo-electron microscopy of extracellular vesicles in fresh plasma

    Directory of Open Access Journals (Sweden)

    Yuana Yuana

    2013-12-01

    Full Text Available Introduction: Extracellular vesicles (EV are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited. Objectives: To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM. Methods: Fresh citrate- and ethylenediaminetetraacetic acid (EDTA-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM. Results: EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles. In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25–260 nm. The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET was employed to determine the 3D structure of platelet secretory granules. Conclusions: Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

  3. Mercury induces proliferation and reduces cell size in vascular smooth muscle cells through MAPK, oxidative stress and cyclooxygenase-2 pathways

    Energy Technology Data Exchange (ETDEWEB)

    Aguado, Andrea; Galán, María; Zhenyukh, Olha; Wiggers, Giulia A.; Roque, Fernanda R. [Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), 28029, Madrid (Spain); Redondo, Santiago [Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, 28040, Madrid (Spain); Peçanha, Franck [Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), 28029, Madrid (Spain); Martín, Angela [Departamento de Bioquímica, Fisiología y Genética Molecular, Universidad Rey Juan Carlos, 28922, Alcorcón (Spain); Fortuño, Ana [Área de Ciencias Cardiovasculares, Centro de Investigación Médica Aplicada, Universidad de Navarra, 31008, Pamplona (Spain); Cachofeiro, Victoria [Departamento de Fisiología, Facultad de Medicina, Universidad Complutense, 28040, Madrid (Spain); Tejerina, Teresa [Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, 28040, Madrid (Spain); Salaices, Mercedes, E-mail: mercedes.salaices@uam.es [Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ), 28029, Madrid (Spain); and others

    2013-04-15

    MAPK activation, oxidative stress and COX-2 expression. ► Inhibition of MAPK reduces HgCl{sub 2}-induced oxidative stress and COX-2 expression. ► Inhibition of MAPK, oxidative stress and COX-2 restores the altered cell proliferation and size.

  4. SMALL VESICLES, BIG VEHICLES: EXOSOMES.

    Directory of Open Access Journals (Sweden)

    Saiz-Lopez P

    2016-09-01

    Full Text Available Exosomes are small membranous vesicles released by different cell types. Since their discovery, they have evolved from being considered simple vehicles for the liberation of cellular wastes, to become one of the most promising fields in the area of biomedical research, and more specifically in oncology, since the different malignant tumors release exosomes to all biological fluids, being involved in various functions of the neoplastic process. At present, it is possible to study these vesicles by minimally invasive techniques in patients, which approach us to obtain a more detailed diagnosis and prognosis, as well as to the discovery of new antitumoral therapies

  5. Using a Reduced Spot Size for Intensity-Modulated Proton Therapy Potentially Improves Salivary Gland-Sparing in Oropharyngeal Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Water, Tara A. van de, E-mail: t.a.van.de.water@rt.umcg.nl [Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Lomax, Antony J. [Centre for Proton Therapy, Paul Scherrer Institute, Villigen-PSI (Switzerland); Bijl, Hendrik P.; Schilstra, Cornelis [Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Hug, Eugen B. [Centre for Proton Therapy, Paul Scherrer Institute, Villigen-PSI (Switzerland); Langendijk, Johannes A. [Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands)

    2012-02-01

    Purpose: To investigate whether intensity-modulated proton therapy with a reduced spot size (rsIMPT) could further reduce the parotid and submandibular gland dose compared with previously calculated IMPT plans with a larger spot size. In addition, it was investigated whether the obtained dose reductions would theoretically translate into a reduction of normal tissue complication probabilities (NTCPs). Methods: Ten patients with N0 oropharyngeal cancer were included in a comparative treatment planning study. Both IMPT plans delivered simultaneously 70 Gy to the boost planning target volume (PTV) and 54 Gy to the elective nodal PTV. IMPT and rsIMPT used identical three-field beam arrangements. In the IMPT plans, the parotid and submandibular salivary glands were spared as much as possible. rsIMPT plans used identical dose-volume objectives for the parotid glands as those used by the IMPT plans, whereas the objectives for the submandibular glands were tightened further. NTCPs were calculated for salivary dysfunction and xerostomia. Results: Target coverage was similar for both IMPT techniques, whereas rsIMPT clearly improved target conformity. The mean doses in the parotid glands and submandibular glands were significantly lower for three-field rsIMPT (14.7 Gy and 46.9 Gy, respectively) than for three-field IMPT (16.8 Gy and 54.6 Gy, respectively). Hence, rsIMPT significantly reduced the NTCP of patient-rated xerostomia and parotid and contralateral submandibular salivary flow dysfunction (27%, 17%, and 43% respectively) compared with IMPT (39%, 20%, and 79%, respectively). In addition, mean dose values in the sublingual glands, the soft palate and oral cavity were also decreased. Obtained dose and NTCP reductions varied per patient. Conclusions: rsIMPT improved sparing of the salivary glands and reduced NTCP for xerostomia and parotid and submandibular salivary dysfunction, while maintaining similar target coverage results. It is expected that rsIMPT improves quality

  6. Vesicles and vesicle fusion: coarse-grained simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2010-01-01

    Biological cells are highly dynamic, and continually move material around their own volume and between their interior and exterior. Much of this transport encapsulates the material inside phospholipid vesicles that shuttle to and fro, fusing with, and budding from, other membranes. A feature of v...

  7. Increased fat mass is associated with increased bone size but reduced volumetric density in pre pubertal children.

    Science.gov (United States)

    Cole, Z A; Harvey, N C; Kim, M; Ntani, G; Robinson, S M; Inskip, H M; Godfrey, K M; Cooper, C; Dennison, E M

    2012-02-01

    Recent studies have shown that obesity is associated with an increased risk of fracture in both adults and children. It has been suggested that, despite greater bone size, obese individuals may have reduced true volumetric density; however this is difficult to assess using two dimensional techniques such as DXA. We evaluated the relationship between fat mass, and bone size and density, in a population cohort of children in whom DXA and pQCT measurements had been acquired. We recruited 530 children at 6 years old from the Southampton Women's Survey. The children underwent measurement of bone mass at the whole body, lumbar spine and hip, together with body composition, by DXA (Hologic Discovery, Hologic Inc., Bedford, MA, USA). In addition 132 of these children underwent pQCT measurements at the tibia (Stratec XCT2000, Stratec Biomedical Systems, Birkenfeld, Germany). Significant positive associations were observed between total fat mass and both bone area (BA) and bone mineral content (BMC) at the whole body minus head, lumbar spine and hip sites (all pfat mass (adjusted for lean mass) was negatively associated with both trabecular and cortical density (β=-14.6 mg/mm(3) per sd, p=0.003; β=-7.7 mg/mm(3) per sd, p=0.02 respectively). These results suggest that fat mass is negatively associated with volumetric bone density at 6 years old, independent of lean mass, despite positive associations with bone size. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. IDENTIFYING DEFECT SIZE IN TWO DIMENSIONAL PLATES BASED ON BOUNDARY MEASUREMENTS USING REDUCED MODEL AND GENETIC ALGORITHM

    Directory of Open Access Journals (Sweden)

    BRAHIM BENAISSA

    2014-12-01

    Full Text Available ­In this study the proper orthogonal decomposition method is utilised as a model reduction technique in crack size estimation in a cracked plate under traction problem. The idea is to create a reduced model based on the results issued from finite element method, thus the crack size parameter is directly related to the boundary displacement obtained from the boundary nodes considered as sensor points. The inverse investigation is run using a genetic algorithm to minimization the error function expressed as the difference between data caused by the crack proposed by genetic algorithm in every individual and the one measured at the actual crack identity. The reduced model is validated by comparing the estimated structural response with the corresponding results from the finite element model. The effectiveness of the approach related to the used number of sensors is presented. Finally the stability of the method against uncertainty is tested by introducing different levels of white noise to the reference data.

  9. Beta(2)-adrenoceptor agonist clenbuterol reduces infarct size and myocardial apoptosis after myocardial ischaemia/reperfusion in anaesthetized rats.

    Science.gov (United States)

    Zhang, Qiufang; Xiang, Jizhou; Wang, Xuanbin; Liu, Hui; Hu, Benrong; Feng, Mei; Fu, Qin

    2010-07-01

    Considerable evidence indicates that the beta(2)-adrenoceptor agonist clenbuterol decreases apoptosis in a rodent model of ischaemic cardiomyopathy. In this study, we investigated the effects of clenbuterol on infarct size caused by myocardial ischaemia/reperfusion (I/R) in anaesthetized rats. Rats were randomly assigned to the following groups: (i) sham (ii) I/R (iii) clenbuterol + I/R (iv) ICI 118551 + clenbuterol + I/R (v) metoprolol + clenbuterol + I/R (vi) metoprolol + I/R (vii) pertussis toxin + clenbuterol + I/R. Under anaesthesia, left anterior descending coronary artery was occluded for 30 min followed by reperfusion for 2 h. Compared with the control I/R group,the clenbuterol (0.5 mg.kg(-1), i.p.) group had reduced infarct size, improved diastolic function and sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity, increased superoxide dismutase activity, and decreased malondialdehyde (MDA) level and LDH, CK release. Clenbuterol increased the phosphorylation of ERK1/2, which resulted in inhibition of myocardial apoptosis as indicated by the reduction of terminal deoxynucleotidyltransferase end labelling-positive staining, Bax/Bcl-2 mRNA and caspase-3 protein expression. The G(i)-protein inhibitor pertussis toxin blocked the clenbuterol-induced improvement in cardiac function and infarct size. Pretreatment with ICI 118551(a selective beta(2)-adrenoceptor antagonist) inhibited the effects of clenbuterol mentioned above. The beta(1)-adrenoceptor agonist metoprolol had similar effects to clenbuterol but failed to reduce MDA and improve SERCA activity. When administered together, metoprolol and clenbuterol did not induce synergistic effects. Clenbuterol pretreatment provides significant cardioprotection against ischaemia/reperfusion injury and this is mediated by the beta(2)-adrenoceptor-G(i)-protein signalling. A combination of the beta(2)-adrenoceptor agonist clenbuterol and the beta(1)-antagonist metoprolol did not lead to a synergistic anti

  10. β2-adrenoceptor agonist clenbuterol reduces infarct size and myocardial apoptosis after myocardial ischaemia/reperfusion in anaesthetized rats

    Science.gov (United States)

    Zhang, Qiufang; Xiang, Jizhou; Wang, Xuanbin; Liu, Hui; Hu, Benrong; Feng, Mei; Fu, Qin

    2010-01-01

    Background and purpose: Considerable evidence indicates that the β2-adrenoceptor agonist clenbuterol decreases apoptosis in a rodent model of ischaemic cardiomyopathy. In this study, we investigated the effects of clenbuterol on infarct size caused by myocardial ischaemia/reperfusion (I/R) in anaesthetized rats. Experimental approach: Rats were randomly assigned to the following groups: (i) sham (ii) I/R (iii) clenbuterol + I/R (iv) ICI 118551 + clenbuterol + I/R (v) metoprolol + clenbuterol + I/R (vi) metoprolol + I/R (vii) pertussis toxin + clenbuterol + I/R. Under anaesthesia, left anterior descending coronary artery was occluded for 30 min followed by reperfusion for 2 h. Key results: Compared with the control I/R group,the clenbuterol (0.5 mg·kg−1, i.p.) group had reduced infarct size, improved diastolic function and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity, increased superoxide dismutase activity, and decreased malondialdehyde (MDA) level and LDH, CK release. Clenbuterol increased the phosphorylation of ERK1/2, which resulted in inhibition of myocardial apoptosis as indicated by the reduction of terminal deoxynucleotidyltransferase end labelling-positive staining, Bax/Bcl-2 mRNA and caspase-3 protein expression. The Gi-protein inhibitor pertussis toxin blocked the clenbuterol-induced improvement in cardiac function and infarct size. Pretreatment with ICI 118551(a selective β2-adrenoceptor antagonist) inhibited the effects of clenbuterol mentioned above. The β1-adrenoceptor agonist metoprolol had similar effects to clenbuterol but failed to reduce MDA and improve SERCA activity. When administered together, metoprolol and clenbuterol did not induce synergistic effects. Conclusions and implications: Clenbuterol pretreatment provides significant cardioprotection against ischaemia/reperfusion injury and this is mediated by the β2-adrenoceptor–Gi-protein signalling. A combination of the β2-adrenoceptor agonist clenbuterol and the

  11. Ca2+ Dependence of Synaptic Vesicle Endocytosis.

    Science.gov (United States)

    Leitz, Jeremy; Kavalali, Ege T

    2016-10-01

    Ca(2+)-dependent synaptic vesicle recycling is essential for structural homeostasis of synapses and maintenance of neurotransmission. Although, the executive role of intrasynaptic Ca(2+) transients in synaptic vesicle exocytosis is well established, identifying the exact role of Ca(2+) in endocytosis has been difficult. In some studies, Ca(2+) has been suggested as an essential trigger required to initiate synaptic vesicle retrieval, whereas others manipulating synaptic Ca(2+) concentrations reported a modulatory role for Ca(2+) leading to inhibition or acceleration of endocytosis. Molecular studies of synaptic vesicle endocytosis, on the other hand, have consistently focused on the roles of Ca(2+)-calmodulin dependent phosphatase calcineurin and synaptic vesicle protein synaptotagmin as potential Ca(2+) sensors for endocytosis. Most studies probing the role of Ca(2+) in endocytosis have relied on measurements of synaptic vesicle retrieval after strong stimulation. Strong stimulation paradigms elicit fusion and retrieval of multiple synaptic vesicles and therefore can be affected by several factors besides the kinetics and duration of Ca(2+) signals that include the number of exocytosed vesicles and accumulation of released neurotransmitters thus altering fusion and retrieval processes indirectly via retrograde signaling. Studies monitoring single synaptic vesicle endocytosis may help resolve this conundrum as in these settings the impact of Ca(2+) on synaptic fusion probability can be uncoupled from its putative role on synaptic vesicle retrieval. Future experiments using these single vesicle approaches will help dissect the specific role(s) of Ca(2+) and its sensors in synaptic vesicle endocytosis. © The Author(s) 2015.

  12. Extracellular vesicles in physiological and pathological conditions

    NARCIS (Netherlands)

    Yuana, Yuana; Sturk, Auguste; Nieuwland, Rienk

    2013-01-01

    Body fluids contain surprising numbers of cell-derived vesicles which are now thought to contribute to both physiology and pathology. Tools to improve the detection of vesicles are being developed and clinical applications using vesicles for diagnosis, prognosis, and therapy are under investigation.

  13. Membrane Trafficking and Vesicle Fusion

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 19; Issue 5. Membrane Trafficking and Vesicle Fusion: Post-Palade Era Researchers Win the Nobel Prize. Riddhi Atul Jani Subba Rao Gangi Setty. General Article Volume 19 Issue 5 May 2014 pp 421-445 ...

  14. Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus.

    Science.gov (United States)

    Chanda, Anindya; Roze, Ludmila V; Pastor, Alicia; Frame, Melinda K; Linz, John E

    2009-07-01

    Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36 h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a "one-step high density cushion". The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts.

  15. A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk

    DEFF Research Database (Denmark)

    Blans, Kristine; Larsen, Lotte Bach; Wiking, Lars

    2014-01-01

    Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known...... components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre...... fraction can be obtained from skim milk by ultracentrifugation. Casein micelle remnants as well as smaller protein components in the crude vesicle fraction can be successfully removed by size chromatography. Electron microscopy of the vesicle isolate reveals circular structures with membrane vesicle...

  16. Complex dynamics of a bilamellar vesicle as a simple model for leukocytes

    Science.gov (United States)

    Kaoui, Badr; Krüger, Timm; Harting, Jens

    The influence of the internal structure of a biological cell (e.g., a leukocyte) on its dynamics and rheology is not yet fully understood. By using 2D numerical simulations of a bilamellar vesicle (BLV) consisting of two vesicles as a cell model, we find that increasing the size of the inner vesicle (mimicking the nucleus) triggers a tank-treading-to-tumbling transition. A new dynamical state is observed, the undulating motion: the BLV inclination with respect to the imposed flow oscillates while the outer vesicle develops rotating lobes. The BLV exhibits a non-Newtonian behavior with a time-dependent apparent viscosity during its unsteady motion. Depending on its inclination and on its inner vesicle dynamical state, the BLV behaves like a solid or a liquid.

  17. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids

    Science.gov (United States)

    Taylor, Douglas D.; Gercel-Taylor, Cicek

    2013-01-01

    We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring. PMID:23908664

  18. Differential regulation of synaptic vesicle tethering and docking by UNC-18 and TOM-1

    Directory of Open Access Journals (Sweden)

    Elena O Gracheva

    2010-10-01

    Full Text Available The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18, unc-64(syntaxin and tom-1(tomosyn. We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin

  19. Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs

    Energy Technology Data Exchange (ETDEWEB)

    Nieh, Mu-Ping [Univ. of Connecticut, Storrs, CT (United States). Inst. of Materials Science, Dept. of Chemical, Materials & Biomolecular Engineering; Dolinar, Paul [Univ. of Ottawa, ON (Canada); Kucerka, Norbert [National Research Council, Chalk River, ON (Canada). Chalk River Lab., Canadian Neutron Beam Centre; Comenius Univ., Bratislava (Slovakia). Dept. of Physical Chemistry of Drugs; Kline, Steven R. [National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States); Debeer-Schmitt, Lisa M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Littrell, Kenneth C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Katsaras, John [National Research Council, Chalk River, ON (Canada). Chalk River Lab., Canadian Neutron Beam Centre; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Brock Univ., St. Catharines, ON (Canada). Dept. of Physics; Univ. of Guelph, ON (Canada). Guelph-Waterloo Physics Inst.

    2011-09-27

    Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical

  20. Piccolo Promotes Vesicle Replenishment at a Fast Central Auditory Synapse

    Directory of Open Access Journals (Sweden)

    Tanvi Butola

    2017-10-01

    Full Text Available Piccolo and Bassoon are the two largest cytomatrix of the active zone (CAZ proteins involved in scaffolding and regulating neurotransmitter release at presynaptic active zones (AZs, but have long been discussed as being functionally redundant. We employed genetic manipulation to bring forth and segregate the role of Piccolo from that of Bassoon at central auditory synapses of the cochlear nucleus—the endbulbs of Held. These synapses specialize in high frequency synaptic transmission, ideally poised to reveal even subtle deficits in the regulation of neurotransmitter release upon molecular perturbation. Combining semi-quantitative immunohistochemistry, electron microscopy, and in vitro and in vivo electrophysiology we first studied signal transmission in Piccolo-deficient mice. Our analysis was not confounded by a cochlear deficit, as a short isoform of Piccolo (“Piccolino” present at the upstream ribbon synapses of cochlear inner hair cells (IHC, is unaffected by the mutation. Disruption of Piccolo increased the abundance of Bassoon at the AZs of endbulbs, while that of RIM1 was reduced and other CAZ proteins remained unaltered. Presynaptic fiber stimulation revealed smaller amplitude of the evoked excitatory postsynaptic currents (eEPSC, while eEPSC kinetics as well as miniature EPSCs (mEPSCs remained unchanged. Cumulative analysis of eEPSC trains indicated that the reduced eEPSC amplitude of Piccolo-deficient endbulb synapses is primarily due to a reduced readily releasable pool (RRP of synaptic vesicles (SV, as was corroborated by a reduction of vesicles at the AZ found on an ultrastructural level. Release probability seemed largely unaltered. Recovery from short-term depression was slowed. We then performed a physiological analysis of endbulb synapses from mice which, in addition to Piccolo deficiency, lacked one functional allele of the Bassoon gene. Analysis of the double-mutant endbulbs revealed an increase in release probability

  1. Characterization of an additional articular cartilage vesicle fraction that generates calcium pyrophosphate dihydrate crystals in vitro.

    Science.gov (United States)

    Derfus, B; Steinberg, M; Mandel, N; Buday, M; Daft, L; Ryan, L

    1995-08-01

    We previously identified a unique fraction of porcine articular cartilage vesicles, sedimentable at 8 x 10(6) g/min, which generate calcium pyrophosphate dihydrate crystals (CPPD) in vitro. We sought to identify and characterize other fractions of articular cartilage digest, sedimentable at lower g forces, which may also contain mineralizing vesicles. Electron microscopy and alkaline phosphatase and nucleoside triphosphate pyrophosphohydrolase (NTPPPH) assays were used to analyze each fraction. Radiometric mineralization assays, Fourier transform infrared (FTIR) spectroscopy, and compensated polarized light microscopy were used to analyze crystals formed by these fractions. Vesicles of varying sizes identical to epiphyseal cartilage matrix vesicles were seen in all sedimentable fractions examined, but were the exclusive component of fractions sedimentable at 3 x 10(6) g/min, termed the heavy vesicle fraction (HVF), and at 8 x 10(6) g/min, now termed the light vesicle fraction (LVF). All vesicle containing fractions supported ATP dependent calcium pyrophosphate precipitation. The HVF and LVF precipitated 30 x more calcium than vesicle poor supernatant (p < 0.01) and 1.5-4 x more than cell-free unfractionated digest (p < 0.01). HVF differed from LVF in that it contained 3-4 x higher NTPPPH specific activity (p < 0.05). HVF resembled LVF in that both precipitated crystals consistent with CPPD by FTIR spectroscopy and compensated polarized light microscopy. These data expand our previous estimate of the total number of vesicles available for biologic mineralization and demonstrate heterogeneity of vesicle fractions. They support a key role for vesicles in CPPD crystal formation.

  2. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells.

    Science.gov (United States)

    Castellano-Muñoz, Manuel; Schnee, Michael E; Ricci, Anthony J

    2016-01-01

    Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking. Copyright © 2016 the American Physiological Society.

  3. NCX4016 (NO-aspirin) reduces infarct size and suppresses arrhythmias following myocardial ischaemia/reperfusion in pigs

    Science.gov (United States)

    Wainwright, Cherry L; Miller, Ashley M; Work, Lorraine M; Soldato, Piero Del

    2002-01-01

    The effect of the nitro-derivative of aspirin, NCX4016, was assessed on ischaemic ventricular arrhythmias and myocardial infarct size in anaesthetized pigs in comparison to native aspirin.Pigs were given aspirin (10 mg kg−1; n=6), low dose NCX4016 (18.4 mg kg−1; n=6) or high dose NCX4016 (60 mg kg−1; n=7) orally for 5 days prior to coronary occlusion and reperfusion. None of the interventions had any effect on baseline haemodynamics prior to coronary occlusion in comparison to control pigs (n=9). Aspirin and high dose NCX4016 both prevented the generation of thromboxane A2 from platelets activated ex vivo with A23187 (30 μM), whereas all three interventions markedly attenuated platelet aggregation in response to collagen in whole blood in comparison to controls.None of the drug interventions had any effect on the incidence of ventricular fibrillation (VF) during myocardial ischaemia (100% in all groups). However, 60 mg kg−1 NCX4016 significantly attenuated the total number of premature ventricular beats (PVB's) (62±16 vs 273±40 in control pigs; Pmyocardial infarct size (22.6±3.7% of area at risk vs 53.0±2.8% of area at risk in control pigs; Paspirin, NCX4016, is an effective antiplatelet agent, which unlike aspirin also reduces the extent of myocardial injury following ischaemia and reperfusion. PMID:11959790

  4. A morphometric study of Reye's syndrome. Correlation of reduced mitochondrial numbers and increased mitochondrial size with clinical manifestations.

    Science.gov (United States)

    Daugherty, C. C.; Gartside, P. S.; Heubi, J. E.; Saalfeld, K.; Snyder, J.

    1987-01-01

    Morphometric analysis of liver ultrastructure in 14 children with Reye's syndrome (RS) of varying morphologic severity was compared with that of 6 children with normal livers. Results showed reduced numbers of enlarged mitochondria in RS. Multivariant analysis identified correlations between increased mitochondrial size, decreased mitochondrial number, and severity of neurologic disease (stage). A disproportionate increase in mitochondrial area and perimeter in the RS cases with the most depressed mitochondrial number distinguished the 4 children with residual neurologic damage or death. Serum salicylate concentrations were negatively correlated with severity of morphologic alteration. Two cases of non-RS salicylate toxicity showed normal or near-normal mitochondrial size and number. In fatty liver from an autopsy specimen from a child, a child with carnitine deficiency, and a child on therapy for dermatomyositis, mitochondrial numbers were also normal. Decreased mitochondrial numbers are characteristic of RS and imply a pathophysiologic mechanism, possibly related to impaired mitochondrial replication. Synergism with other forms of mitochondrial injury, such as salicylates, hypoglycemia, or shock may influence clinical severity, progression, and outcome. Images Figure 1 Figure 5 Figure 6 PMID:2823614

  5. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Yokokawa, Takumi; Endo, Yuriko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Iwanaka, Nobumasa [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Higashida, Kazuhiko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Faculty of Sport Science, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192 (Japan); Taguchi, Sadayoshi [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)

    2013-10-11

    Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

  6. Role of extracellular vesicles in rheumatoid arthritis.

    Science.gov (United States)

    Fu, Haitao; Hu, Die; Zhang, Licheng; Tang, Peifu

    2018-01-01

    Cell-derived extracellular vesicles (EVs) are involved in the pathogenesis of rheumatoid arthritis (RA), playing important roles in antigen presentation, inflammation, angiogenesis, cell-cell signal communication, thrombosis, and articular cartilage extracellular matrix degradation. Understanding the pathogenic mechanism of RA is important for developing therapies. The pathogenic indicators of RA, such as submicron-sized EVs, represent promising biomarkers for evaluating RA activity. This review summarizes the recent advances in understanding the pathogenesis of RA, and sheds light on the pathogenic as well as anti-inflammatory or immunosuppressive roles of EVs. We suggest that EVs could be harnessed as tools for drug delivery or targets for RA therapies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. A New Technique for Reducing Size of a WPT System Using Two-Loop Strongly-Resonant Inductors

    Directory of Open Access Journals (Sweden)

    Matjaz Rozman

    2017-10-01

    Full Text Available Mid-range resonant coupling-based high efficient wireless power transfer (WPT techniques have gained substantial research interest due to the number of potential applications in many industries. This paper presents a novel design of a resonant two-loop WPT technique including the design, fabrication and preliminary results of this proposal. This new design employs a compensation inductor which is combined with the transmitter and receiver loops in order to significantly scale down the size of the transmitter and receiver coils. This can improve the portability of the WPT transmitters in practical systems. Moreover, the benefits of the system enhancement are not only limited to the lessened magnitude of the T X & R X , simultaneously both the weight and the bill of materials are also minimised. The proposed system also demonstrates compatibility with the conventional electronic components such as capacitors hence the development of the T X & R X is simplified. The proposed system performance has been validated using the similarities between the experimental and simulation results. The power efficiency of the prototype circuit is found to be 93%, which is close to the efficiency reached by the conventional design. However, the weight of the transmitter and receiver inductors is now reduced by 78%, while the length of these inductors is reduced by 80%.

  8. A natural antipredation experiment: predator control and reduced sea ice increases colony size in a long-lived duck.

    Science.gov (United States)

    Hanssen, Sveinn A; Moe, Børge; Bårdsen, Bård-Jørgen; Hanssen, Frank; Gabrielsen, Geir W

    2013-09-01

    Anthropogenic impact on the environment and wildlife are multifaceted and far-reaching. On a smaller scale, controlling for predators has been increasing the yield from local natural prey resources. Globally, human-induced global warming is expected to impose severe negative effects on ecosystems, an effect that is expected to be even more pronounced in the scarcely populated northern latitudes. The clearest indication of a changing Arctic climate is an increase in both air and ocean temperatures leading to reduced sea ice distribution. Population viability is for long-lived species dependent on adult survival and recruitment. Predation is the main mortality cause in many bird populations, and egg predation is considered the main cause of reproductive failure in many birds. To assess the effect of predation and climate, we compared population time series from a natural experiment where a trapper/down collector has been licensed to actively protect breeding common eiders Somateria mollissima (a large seaduck) by shooting/chasing egg predators, with time series from another eider colony located within a nature reserve with no manipulation of egg predators. We found that actively limiting predator activity led to an increase in the population growth rate and carrying capacity with a factor of 3-4 compared to that found in the control population. We also found that population numbers were higher in years with reduced concentration of spring sea ice. We conclude that there was a large positive impact of human limitation of egg predators, and that this lead to higher population growth rate and a large increase in size of the breeding colony. We also report a positive effect of warming climate in the high arctic as reduced sea-ice concentrations was associated with higher numbers of breeding birds.

  9. Phospholipid Vesicles in Materials Science

    Energy Technology Data Exchange (ETDEWEB)

    Granick, Steve [Univ. of Illinois, Champaign, IL (United States)

    2016-05-11

    The objective of this research was to develop the science basis needed to deploy phospholipid vesicles as functional materials in energy contexts. Specifically, we sought to: (1) Develop an integrated molecular-level understanding of what determines their dynamical shape, spatial organization, and responsiveness to complex, time-varying environments; and (2) Develop understanding of their active transportation in crowded environments, which our preliminary measurements in cells suggest may hold design principles for targeting improved energy efficiency in new materials systems. The methods to do this largely involved fluorescence imaging and other spectroscopy involving single particles, vesicles, particles, DNA, and endosomes. An unexpected importance outcome was a new method to image light-emitting diodes during actual operation using super-resolution spectroscopy.

  10. Dynamics of endocytic vesicle creation.

    Science.gov (United States)

    Perrais, David; Merrifield, Christien J

    2005-11-01

    Clathrin-mediated endocytosis is the main path for receptor internalization in metazoans and is essential for controlling cell integrity and signaling. It is driven by a large array of protein and lipid interactions that have been deciphered mainly by biochemical and genetic means. To place these interactions into context, and ultimately build a fully operative model of endocytosis at the molecular level, it is necessary to know the kinetic details of the role of each protein in this process. In this review, we describe the recent efforts made, by using live cell imaging, to define clear steps in the formation of endocytic vesicles and to observe the recruitment of key proteins during membrane invagination, the scission of a newly formed vesicle, and its movement away from the plasma membrane.

  11. Extracellular vesicles and blood diseases.

    Science.gov (United States)

    Nomura, Shosaku

    2017-04-01

    Extracellular vesicles (EVs) are small membrane vesicles released from many different cell types by the exocytic budding of the plasma membrane in response to cellular activation or apoptosis. EVs disseminate various bioactive effectors originating from the parent cells and transfer functional RNA and protein between cells, enabling them to alter vascular function and induce biological responses involved in vascular homeostasis. Although most EVs in human blood originate from platelets, EVs are also released from leukocytes, erythrocytes, endothelial cells, smooth muscle cells, and cancer cells. EVs were initially thought to be small particles with procoagulant activity; however, they can also evoke cellular responses in the immediate microenvironments and transport microRNAs (miRNA) into target cells. In this review, we summarize the recent literature relevant to EVs, including a growing list of clinical disorders that are associated with elevated EV levels. These studies suggest that EVs play roles in various blood diseases.

  12. Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Brisson, Alain R

    2017-01-01

    Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

  13. Pharmacologic myocardial preconditioning with monophosphoryl lipid A (MLA) reduces infarct size and stunning in dogs and rabbits.

    Science.gov (United States)

    Elliott, G T

    1996-09-30

    As the mechanism of ischemic preconditioning unfolds, various strategies for inducing pharmacologic preconditioning become apparent. Adenosine receptor agonists, KATP channel activators, and endothelial-neutrophil adhesion antagonists have enjoyed cardioprotective activity against ischemia/reperfusion injury in at least some preclinical models. Monophosphoryl lipid A (MLA), a structural derivative of the pharmacophore of endotoxin, enjoys an improved therapeutic index in relation to the parent biological product. MLA has found clinical application as a vaccine adjuvant and protects from sepsis and septic shock in the preclinical setting. In animal models of myocardial ischemia/reperfusion injury, pretreatment 12-24 hours prior to ischemia with a single IV bolus injection of MLA limits infarct size 50 to 75 percent in standard canine and rabbit models at doses of 10-35 micrograms/kg. Regional myocardial stunning following multiple 5-minute ischemic episodes as assessed by segment shortening is reduced in dogs pretreated 24 but not 1 hour prior to ischemia. Global cardiac function, as evaluated by pressure-volume constructs generated in dogs being weaned from cardiopulmonary bypass, recovers more quickly in animals pretreated with MLA. Cardiac protection in various models is associated with preservation of ATP during ischemia, induction of 5' nucleotidase and enhancement of calcium reuptake by SR during reperfusion. Limitation of infarct size by MLA in dogs and rabbits can be reversed by the administration of glibenclamide just prior to ischemia, suggesting a role for KATP channel opening during the first minutes of sustained ischemia. A clinical formulation of MLA (MPL-C) is currently undergoing clinical investigation in the Phase II setting in coronary artery bypass surgical patients. MLA may represent a novel means of inducing pharmacologic preconditioning, with potential for clinical application as a pretreatment before planned myocardial ischemia.

  14. Polymeric surfactant vesicles. Synthesis and characterization by nuclear magnetic resonance spectroscopy and dynamic laser light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Kippenberger, D. (Texas A and M Univ., College Station); Rosenquist, K.; Odberg L.; Tundo, P.; Fendler, J.H.

    1983-03-09

    (CH/sub 3/(CH/sub 2/)/sub 15/)(CH/sub 2/==CH(CH/sub 2/)/sub 8/CONH(CH/sub 2/)/sub 6/)(CH/sub 3/)/sub 2/N/sup +/Br/sup -/(1),(CH/sub 3/(CH/sub 2/)/sub 14/CO/sub 2/(CH/sub 2/)/sub 2/)/sub 2/(CH/sub 3/)(CH/sub 2/CH==CH/sub 2/)N/sup +/Br/sup -/(2),(CH/sub 3/(CH/sub 2/)/sub 14/CO/sub 2/(CH/sub 2/)/sub 2/)/sub 2/NCOCH==CHCO/sub 2/H(3), and (CH/sub 3/(CH/sub 2/)/sub 17/)NCOCH==CHCO/sub 2/H(4) have been synthesized. Sonication of these surfactants led to the formation of vesicles. Vesicles could be polymerized by exposure to ultraviolet radiation or by the use of azoisobutyronitrile (AIBN) as an initiator. Vesicles prepared from 1 polymerized in their bilayers. Vesicles prepared from 2, 3, and 4 have double bonds on their headgroups and could, therefore, potentially polymerize both at the inner and outer surfaces or separately at either surface. Polymerization of vesicles prepared from 2, 3, and 4 by ultraviolet radiation resulted in the closing of both surfaces. Conversely, addition of AIBN to a solution of these vesicles and subsequent incubation at 80/sup 0/ led to the selective ''zipping-up'' of the outer surface only. Following the loss of vinyl protons of the surfactant vesicles by /sup 1/HNMR spectroscopy provided evidence for polymerization. Presence of vesicles has been demonstrated by substrate entrapment, gel filtration, and dynamic laser light scattering. Increasing the sonication time led to smaller and less polydisperse vesicles. On polymerization, vesicles maintaned the sizes of their nonpolymeric counterparts. Polymeric vesicles were found to be appreciably more stable than their unpolymerized analogues. Polymeric vesicles retained the fluidities of vesicles and underwent thermotropic phase transitions. Addition of KCl led to the growth of both unpolymerized and polymeric surfactant vesicles.

  15. An Examination of Teachers' Perceptions and Practice when Teaching Large and Reduced-Size Classes: Do Teachers Really Teach Them in the Same Way?

    Science.gov (United States)

    Harfitt, Gary James

    2012-01-01

    Class size research suggests that teachers do not vary their teaching strategies when moving from large to smaller classes. This study draws on interviews and classroom observations of three experienced English language teachers working with large and reduced-size classes in Hong Kong secondary schools. Findings from the study point to subtle…

  16. Dimensional characterization of extracellular vesicles using atomic force microscopy

    Science.gov (United States)

    Sebaihi, N.; De Boeck, B.; Yuana, Y.; Nieuwland, R.; Pétry, J.

    2017-03-01

    Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from healthy subjects to patients suffering from particular disease. So, EV have gained a strong scientific and clinical interest as potential biomarkers for diagnosis and prognosis of disease. Due to their small size, accurate detection and characterization of EV remain challenging. The aim of the presented work is to propose a characterization method of erythrocyte-derived EV using atomic force microscopy (AFM). The vesicles are immobilized on anti-CD235a-modified mica and analyzed by AFM under buffer liquid and dry conditions. EV detected under both conditions show very similar sizes namely ~30 nm high and ~90 nm wide. The size of these vesicles remains stable over drying time as long as 7 d at room temperature. Since the detected vesicles are not spherical, EV are characterized by their height and diameter, and not only by the height as is usually done for spherical nanoparticles. In order to obtain an accurate measurement of EV diameters, the geometry of the AFM tip was evaluated to account for the lateral broadening artifact inherent to AFM measurements. To do so, spherical polystyrene (PS) nanobeads and EV were concomitantly deposited on the same mica substrate and simultaneously measured by AFM under dry conditions. By applying this procedure, direct calibration of the AFM tip could be performed together with EV characterization under identical experimental conditions minimizing external sources of uncertainty on the shape and size of the tip, thus allowing standardization of EV measurement.

  17. Enhanced mitochondrial complex gene function and reduced liver size may mediate improved feed efficiency of beef cattle during compensatory growth.

    Science.gov (United States)

    Connor, Erin E; Kahl, Stanislaw; Elsasser, Theodore H; Parker, Joel S; Li, Robert W; Van Tassell, Curtis P; Baldwin, Ransom L; Barao, Scott M

    2010-03-01

    Growing ruminants under extended dietary restriction exhibit compensatory growth upon ad libitum feeding, which is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify mechanisms contributing to these physiological changes, 8-month-old steers were fed either ad libitum (control; n = 6) or 60-70% of intake of control animals (feed-restricted; n = 6) for a period of 12 weeks. All steers were fed ad libitum for the remaining 8 weeks of experimentation (realimentation). Liver was biopsied at days -14, +1, and +14 relative to realimentation for gene expression analysis by microarray hybridization. During early realimentation, feed-restricted steers exhibited greater rates of gain and feed efficiency than controls and an increase in expression of genes functioning in cellular metabolism, cholesterol biosynthesis, oxidative phosphorylation, glycolysis, and gluconeogenesis. Gene expression changes during feed restriction were similar to those reported in mice, indicating similar effects of caloric restriction across species. Based on expression of genes involved in cell division and growth and upregulation of genes encoding mitochondrial complex proteins in early realimentation, it was concluded that reduced hepatic size and increased mitochondrial function may contribute to improved feed efficiency observed during compensatory growth.

  18. A Self-Organizing Map-Based Approach to Generating Reduced-Size, Statistically Similar Climate Datasets

    Science.gov (United States)

    Cabell, R.; Delle Monache, L.; Alessandrini, S.; Rodriguez, L.

    2015-12-01

    Climate-based studies require large amounts of data in order to produce accurate and reliable results. Many of these studies have used 30-plus year data sets in order to produce stable and high-quality results, and as a result, many such data sets are available, generally in the form of global reanalyses. While the analysis of these data lead to high-fidelity results, its processing can be very computationally expensive. This computational burden prevents the utilization of these data sets for certain applications, e.g., when rapid response is needed in crisis management and disaster planning scenarios resulting from release of toxic material in the atmosphere. We have developed a methodology to reduce large climate datasets to more manageable sizes while retaining statistically similar results when used to produce ensembles of possible outcomes. We do this by employing a Self-Organizing Map (SOM) algorithm to analyze general patterns of meteorological fields over a regional domain of interest to produce a small set of "typical days" with which to generate the model ensemble. The SOM algorithm takes as input a set of vectors and generates a 2D map of representative vectors deemed most similar to the input set and to each other. Input predictors are selected that are correlated with the model output, which in our case is an Atmospheric Transport and Dispersion (T&D) model that is highly dependent on surface winds and boundary layer depth. To choose a subset of "typical days," each input day is assigned to its closest SOM map node vector and then ranked by distance. Each node vector is treated as a distribution and days are sampled from them by percentile. Using a 30-node SOM, with sampling every 20th percentile, we have been able to reduce 30 years of the Climate Forecast System Reanalysis (CFSR) data for the month of October to 150 "typical days." To estimate the skill of this approach, the "Measure of Effectiveness" (MOE) metric is used to compare area and overlap

  19. X-ray microanalysis of mineralized matrix vesicles of experimental saccular aneurysms

    Energy Technology Data Exchange (ETDEWEB)

    Greenhill, N.S.; Presland, M.R.; Rogers, K.M.; Stehbens, W.E.

    1985-10-01

    An energy dispersive X-ray microanalytical study was designed to examine the mineral deposits in matrix vesicles found in the walls of experimental aneurysms from two rabbits (103 and 1071 days postoperatively) and two sheep aneurysms (234 and 1202 days postoperatively). The freeze-substitution technique was adopted for use to retain inorganic ions in situ. Numerous, various sized extracellular electron-dense structures, believed to be matrix vesicles were observed. Size histograms for the mineralized vesicles showed that the proportion of smaller vesicles was higher in the older animals. A total of 370 vesicles were analyzed. Calcium and phosphorus with small amounts of magnesium were identified. No particular calcium phosphate mineral was dominant with the mean Ca/P molar ratio for all animals falling in the 1.1-1.2 range. Correlation coefficients for interrelationships between calcium, phosphorus, magnesium, and size were weak except for calcium vs phosphorus which was close to one, consistent with some type of calcium phosphate being the major constituent of the mineralized vesicles. Smaller electron-dense particles, probably mitochondrial granules were seen in some smooth muscle cells; a small number were analyzed and contained calcium and phosphorus (mean Ca/P molar ratio of 0.86) but no magnesium.

  20. Synaptic vesicle proteins and active zone plasticity

    Directory of Open Access Journals (Sweden)

    Robert J Kittel

    2016-04-01

    Full Text Available Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone. The complex molecular architecture of active zones mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of active zones vary significantly, even for a given connection. Thus, there appear to be distinct active zone states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the active zone.The protein-rich cytomatrix at the active zone (CAZ provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1 and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and active zone states, which has heretofore received little attention.

  1. Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles

    NARCIS (Netherlands)

    Farsi, Z.; Preobraschenski, J.; Bogaart, G. van den; Riedel, D.; Jahn, R.; Woehler, A.

    2016-01-01

    Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided

  2. Extracellular Vesicles: Evolving Contributors in Autoimmunity

    OpenAIRE

    Katsiougiannis, Stergios

    2015-01-01

    Extracellular vesicles, including microvesicles, exosomes and apoptotic bodies are recognized as carriers of pathogen-associated molecules with direct involvement in immune signaling and inflammation. Those observations have enforced the way these membranous vesicles are being considered as promising immunotherapeutic targets. In this review, we discuss the emerging roles of extracellular vesicles in autoimmunity and highlights their potential use as disease biomarkers as well as targets for ...

  3. Exosomes: secreted vesicles and intercellular communications

    OpenAIRE

    Théry, Clotilde

    2011-01-01

    Exosomes are small membrane vesicles of endocytic origin secreted by most cell types, and are thought to play important roles in intercellular communications. Although exosomes were originally described in 1983, interest in these vesicles has really increased dramatically in the last 3 years, after the finding that they contain mRNA and microRNA. This discovery sparked renewed interest for the general field of membrane vesicles involved in intercellular communications, and research on these s...

  4. Biological reference materials for extracellular vesicle studies.

    Science.gov (United States)

    Valkonen, S; van der Pol, E; Böing, A; Yuana, Y; Yliperttula, M; Nieuwland, R; Laitinen, S; Siljander, P R M

    2017-02-15

    Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis

    Science.gov (United States)

    Mantri, Chinmay K; Chen, Chin-Ho; Dong, Xinhong; Goodwin, Jeffery Shawn; Pratap, Siddharth; Paromov, Victor; Xie, Hua

    2015-01-01

    Porphyromonas gingivalis is a keystone periopathogen that plays an essential role in the progress of periodontitis. Like other gram-negative bacteria, the ability of P. gingivalis to produce outer membrane vesicles is a strategy used to interact with, and survive within its biological niches. Here we compared the protein components associated with vesicles derived from a fimbriated strain (33277) and an afimbriated strain (W83) of P. gingivalis using proteomic analyses. Some well-known virulence factors were identified in vesicles from both strains, such as gingipains and hemagglutinin. In contrast, FimC, FimD, and FimE, minor components of long fimbriae were found exclusively in 33277 vesicles, while proteins with a tetratricopeptide repeat (TPR) domain were unique to W83 vesicles. We found that significantly more 33277 than W83 vesicles were internalized into human oral keratinocytes and gingival fibroblasts. Interestingly, FimA, a well-known adhesin responsible for the attachment and invasion of P. gingivalis into host cells, was not essential for the invasive capabilities of P. gingivalis vesicles. Rather minor components of long fimbriae were required for an efficient invasive activity of vesicles. The most striking finding was that P. gingivalis strains lacking or having a reduced FimA expression showed a significant reduction in vesiculation. These results suggest that production and pathogenicity of P. gingivalis vesicles may largely depend on expression of the fim locus, and that the integration of vesicle production and pathogenicity with fimbrial expression may allow P. gingivalis to confer upon itself certain functional advantages. PMID:25524808

  6. Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis.

    Science.gov (United States)

    Mantri, Chinmay K; Chen, Chin-Ho; Dong, Xinhong; Goodwin, Jeffery Shawn; Pratap, Siddharth; Paromov, Victor; Xie, Hua

    2015-02-01

    Porphyromonas gingivalis is a keystone periopathogen that plays an essential role in the progress of periodontitis. Like other gram-negative bacteria, the ability of P. gingivalis to produce outer membrane vesicles is a strategy used to interact with, and survive within its biological niches. Here we compared the protein components associated with vesicles derived from a fimbriated strain (33277) and an afimbriated strain (W83) of P. gingivalis using proteomic analyses. Some well-known virulence factors were identified in vesicles from both strains, such as gingipains and hemagglutinin. In contrast, FimC, FimD, and FimE, minor components of long fimbriae were found exclusively in 33277 vesicles, while proteins with a tetratricopeptide repeat (TPR) domain were unique to W83 vesicles. We found that significantly more 33277 than W83 vesicles were internalized into human oral keratinocytes and gingival fibroblasts. Interestingly, FimA, a well-known adhesin responsible for the attachment and invasion of P. gingivalis into host cells, was not essential for the invasive capabilities of P. gingivalis vesicles. Rather minor components of long fimbriae were required for an efficient invasive activity of vesicles. The most striking finding was that P. gingivalis strains lacking or having a reduced FimA expression showed a significant reduction in vesiculation. These results suggest that production and pathogenicity of P. gingivalis vesicles may largely depend on expression of the fim locus, and that the integration of vesicle production and pathogenicity with fimbrial expression may allow P. gingivalis to confer upon itself certain functional advantages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  7. Polymer Vesicles as Robust Scaffolds for the Directed Assembly of Highly Crystalline Nanocrystals †

    KAUST Repository

    Wang, Mingfeng

    2009-12-15

    We report the incorporation of various inorganic nanoparticles (NPs) (PbS, LaOF, LaF3, and TiO2, each capped by oleic acid, and CdSe/ZnS core/shell QDs capped by trioctylphosphine oxide) into vesicles (d = 70-150 nm) formed by a sample of poly(styrene-b-acrylic acid) (PS4o4-b-PAA 62, where the subscripts refer to the degree of polymerization) in mixtures of tetrahydrofuran (THF), dioxane, and water. The block copolymer formed mixtures of crew-cut micelles and vesicles with some enhancement of the vesicle population when the NPs were present. The vesicle fraction could be isolated by selective sedimentation via centrifugation, followed by redispersion in water. The NPs appeared to be incorporated into the PAA layers on the internal and external walls of the vesicles (strongly favoring the former). NPs on the exterior surface of the vesicles could be removed completely by treating the samples with a solution of ethylenediaminetetraacetate (EDTA) in water. The triangular nanoplatelets of LaF3 behaved differently. Stacks of these platelets were incorporated into solid colloidal entities, similar in size to the empty vesicles that accompanied them, during the coassembly as water was added to the polymer/LaF3/THF/ dioxane mixture. © 2009 American Chemical Society.

  8. Toward hybrid proteo-polymeric vesicles generating a photoinduced proton gradient for biofuel cells

    Science.gov (United States)

    Choi, Hyo-Jick; Lee, Hyeseung; Montemagno, Carlo D.

    2005-09-01

    We describe our efforts towards constructing a hybrid protein-polymer vesicle device based on the photoactive protein, bacteriorhodopsin (BR), for applications in the area of biosensors and biofuel cells. Successful protein incorporation into biomimetic polymer vesicles is a prerequisite for developing hybrid 'nano-bio' integrated devices. We suggest a systematic procedure for creating energy transducing, protein-incorporating, functional vesicles, based on the morphological ternary diagram. First, we constructed the morphological ternary diagram of the water/ethanol/polymer system with a size distribution of vesicles. The polymer used was an ABA triblock copolymer, PEtOz-PDMS-PEtOz [poly(2-ethyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-ethyl-2-oxazoline)]. Second, we incorporated BR in the form of purple membrane (PM) into polymer vesicle membranes under several different conditions, based on the morphological ternary diagram. Generation of electrochemical energy by BR proton pumping was checked by monitoring the pH change in parallel with transmission electron microscope analysis. The morphology of the polymer vesicles changed very little with the addition of PM. This work shows that the morphological ternary diagram provides a systematic method for constructing successful hybrid BR-incorporating biomimetic polymer vesicles.

  9. Extracellular vesicles: structure, function, and potential clinical uses in renal diseases

    Directory of Open Access Journals (Sweden)

    F.T. Borges

    2013-10-01

    Full Text Available Interest in the role of extracellular vesicles in various diseases including cancer has been increasing. Extracellular vesicles include microvesicles, exosomes, apoptotic bodies, and argosomes, and are classified by size, content, synthesis, and function. Currently, the best characterized are exosomes and microvesicles. Exosomes are small vesicles (40-100 nm involved in intercellular communication regardless of the distance between them. They are found in various biological fluids such as plasma, serum, and breast milk, and are formed from multivesicular bodies through the inward budding of the endosome membrane. Microvesicles are 100-1000 nm vesicles released from the cell by the outward budding of the plasma membrane. The therapeutic potential of extracellular vesicles is very broad, with applications including a route of drug delivery and as biomarkers for diagnosis. Extracellular vesicles extracted from stem cells may be used for treatment of many diseases including kidney diseases. This review highlights mechanisms of synthesis and function, and the potential uses of well-characterized extracellular vesicles, mainly exosomes, with a special focus on renal functions and diseases.

  10. Trafficking of astrocytic vesicles in hippocampal slices

    Energy Technology Data Exchange (ETDEWEB)

    Potokar, Maja; Kreft, Marko [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia); Lee, So-Young; Takano, Hajime; Haydon, Philip G. [Department of Neuroscience, Room 215, Stemmler Hall, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104 (United States); Zorec, Robert, E-mail: Robert.Zorec@mf.uni-lj.si [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia)

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  11. Extracellular vesicles in cartilage homeostasis and osteoarthritis.

    Science.gov (United States)

    Miyaki, Shigeru; Lotz, Martin K

    2018-01-01

    Extracellular vesicles carry bioactive molecules that can be transferred between cells and tissues. The purpose of this review is to describe how extracellular vesicles regulate functions of cells in cartilage and other joint tissues. The potential application of extracellular vesicles in the treatment of osteoarthritis and as biomarkers will also be discussed. Extracellular vesicles are found in synovial fluid, in articular cartilage and in the supernatants of synoviocytes and chondrocytes. Extracellular vesicles in cartilage have been proposed to be involved in cross talk between cells in joint tissues and to affect extracellular matrix turnover and inflammation. Extracellular vesicles from arthritic joints can promote abnormal gene expression and changes in cartilage extracellular matrix, including abnormal mineralization. Promising results were obtained in the therapeutic application of mesenchymal stem cell-derived extracellular vesicles for cartilage repair and experimental osteoarthritis. Extracellular vesicles have emerged as vehicles for the exchange of bioactive signaling molecules within cartilage and between joint tissues to promote joint homeostasis and arthritis pathogenesis. As the molecular content of extracellular vesicles can be customized, they offer utility in therapeutic applications.

  12. Cell-derived vesicles exposing coagulant tissue factor in saliva.

    Science.gov (United States)

    Berckmans, René J; Sturk, Auguste; van Tienen, Laurens M; Schaap, Marianne C L; Nieuwland, Rienk

    2011-03-17

    On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.

  13. Transmembrane topology of the acetylcholine receptor examined in reconstituted vesicles

    Energy Technology Data Exchange (ETDEWEB)

    McCrea, P.D.

    1987-01-01

    Each of the five acetylcholine receptor (AChR) subunits, ..cap alpha../sub 2/..beta..-..gamma..delta, is believed to have the same number of transmembrane crossing and to share the same general folding pattern. AChR isolated from the electric organ of electric fish is predominantly dimeric. We have used this bridge as a marker for the C-terminus of the delta subunit, and presumably that of the other subunits in addition. The disulfide's accessibility to hydrophilic reductants, principally glutathione (GSH), was tested in a reconstituted vesicle system. The reduction of the delta-delta desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of delta/sub 2/ to delta was followed by employing non-reducing SDS-PAGE. Reductants such as GSH were able to access the bridge in intact right-side-out vesicles. No acceleration of this process was evident when the vesicles were disrupted by freeze-thaw or by detergents. Control experiments which determined the rate of reduction of entrapped diphtheria toxin, or that of /sup 3/H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space.

  14. Polymeric vesicles: from drug carriers to nanoreactors and artificial organelles.

    Science.gov (United States)

    Tanner, Pascal; Baumann, Patric; Enea, Ramona; Onaca, Ozana; Palivan, Cornelia; Meier, Wolfgang

    2011-10-18

    One strategy in modern medicine is the development of new platforms that combine multifunctional compounds with stable, safe carriers in patient-oriented therapeutic strategies. The simultaneous detection and treatment of pathological events through interactions manipulated at the molecular level offer treatment strategies that can decrease side effects resulting from conventional therapeutic approaches. Several types of nanocarriers have been proposed for biomedical purposes, including inorganic nanoparticles, lipid aggregates, including liposomes, and synthetic polymeric systems, such as vesicles, micelles, or nanotubes. Polymeric vesicles--structures similar to lipid vesicles but created using synthetic block copolymers--represent an excellent candidate for new nanocarriers for medical applications. These structures are more stable than liposomes but retain their low immunogenicity. Significant efforts have been made to improve the size, membrane flexibility, and permeability of polymeric vesicles and to enhance their target specificity. The optimization of these properties will allow researchers to design smart compartments that can co-encapsulate sensitive molecules, such as RNA, enzymes, and proteins, and their membranes allow insertion of membrane proteins rather than simply serving as passive carriers. In this Account, we illustrate the advances that are shifting these molecular systems from simple polymeric carriers to smart-complex protein-polymer assemblies, such as nanoreactors or synthetic organelles. Polymeric vesicles generated by the self-assembly of amphiphilic copolymers (polymersomes) offer the advantage of simultaneous encapsulation of hydrophilic compounds in their aqueous cavities and the insertion of fragile, hydrophobic compounds in their membranes. This strategy has permitted us and others to design and develop new systems such as nanoreactors and artificial organelles in which active compounds are simultaneously protected and allowed to

  15. Allocating Sample Sizes to Reduce Budget for Fixed-Effect 2×2 Heterogeneous Analysis of Variance

    Science.gov (United States)

    Luh, Wei-Ming; Guo, Jiin-Huarng

    2016-01-01

    This article discusses the sample size requirements for the interaction, row, and column effects, respectively, by forming a linear contrast for a 2×2 factorial design for fixed-effects heterogeneous analysis of variance. The proposed method uses the Welch t test and its corresponding degrees of freedom to calculate the final sample size in a…

  16. Extracellular Vesicles in Cardiovascular Theranostics.

    Science.gov (United States)

    Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro, Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

  17. Emergent Properties of Giant Vesicles Formed by a Polymerization-Induced Self-Assembly (PISA) Reaction

    Science.gov (United States)

    Albertsen, Anders N.; Szymański, Jan K.; Pérez-Mercader, Juan

    2017-01-01

    Giant micrometer sized vesicles are of obvious interest to the natural sciences as well as engineering, having potential application in fields ranging from drug delivery to synthetic biology. Their formation often requires elaborate experimental techniques and attempts to obtain giant vesicles from chemical media in a one-pot fashion have so far led to much smaller nanoscale structures. Here we show that a tailored medium undergoing controlled radical polymerization is capable of forming giant polymer vesicles. Using a protocol which allows for an aqueous reaction under mild conditions, we observe the macroscale consequences of amphiphilic polymer synthesis and the resulting molecular self-assembly using fluorescence microscopy. The polymerization process is photoinitiated by blue light granting complete control of the reaction, including on the microscope stage. The self-assembly process leads to giant vesicles with radii larger than 10 microns, exhibiting several emergent properties, including periodic growth and collapse as well as phototaxis.

  18. EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research.

    Science.gov (United States)

    Kim, Dae-Kyum; Lee, Jaewook; Simpson, Richard J; Lötvall, Jan; Gho, Yong Song

    2015-04-01

    For cell-to-cell communication, all living cells including archaea, bacteria, and eukaryotes secrete nano-sized membrane vesicles into the extracellular space. These extracellular vesicles harbor specific subsets of proteins, mRNAs, miRNAs, lipids, and metabolites that represent their cellular status. These vesicle-specific cargos are considered as novel diagnostic biomarkers as well as therapeutic targets. With the advancement in high-throughput technologies on multiomics studies and improvements in bioinformatics approaches, a huge number of vesicular proteins, mRNAs, miRNAs, lipids, and metabolites have been identified, and our understanding of these complex extracellular organelles has considerably increased during these past years. In this review, we highlight EVpedia (http://evpedia.info), a community web portal for systematic analyses of prokaryotic and eukaryotic extracellular vesicles research. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Dynamic light scattering for the characterization and counting of extracellular vesicles: a powerful noninvasive tool

    Science.gov (United States)

    Palmieri, Valentina; Lucchetti, Donatella; Gatto, Ilaria; Maiorana, Alessandro; Marcantoni, Margherita; Maulucci, Giuseppe; Papi, Massimiliano; Pola, Roberto; De Spirito, Marco; Sgambato, Alessandro

    2014-09-01

    Extracellular vesicles (EVs) are cell-to-cell shuttles that have recently drawn interest both as drug delivery platforms and disease biomarkers. Despite the increasingly recognized relevance of these vesicles, their detection, and characterization still have several technical drawbacks. In this paper, we accurately assess the size distribution and concentration of EVs by using a high-throughput non-perturbative technique such as Dynamic Light Scattering (DLS). The vesicle radii distribution, as further confirmed by Atomic Force Microscopy experiments, ranges from 10 to 80 nm and appears very asymmetric towards larger radii with a main peak at roughly 30 nm. By combining DLS and Bradford assay, we also demonstrate the feasibility of recovering the concentration and its distribution of proteins contained inside vesicles. The sensitivity of our approach allows to detect protein concentrations as low as 0.01 mg/ml.

  20. Reconstitution of cytochrome c oxidase in phospholipid vesicles containing polyvinylic polymers.

    Science.gov (United States)

    Sarti, P; Antonini, G; Malatesta, F; Vallone, B; Villaschi, S; Brunori, M; Hider, R C; Hamed, K

    1989-01-01

    Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368]. Images Fig. 2. PMID:2539096

  1. Significance of Extracellular Vesicles: Pathobiological Roles in Disease.

    Science.gov (United States)

    Yamamoto, Seiji; Azuma, Erika; Muramatsu, Masashi; Hamashima, Takeru; Ishii, Yoko; Sasahara, Masakiyo

    2016-11-25

    Over the past decade, many studies have been conducted on extracellular vesicles (EVs) in the fields of basic and clinical research. EVs are small sized membranous vesicles generated from many type of cells upon activation by environmental stresses such as heat, hypoxia, and irradiation. EVs theoretically consist of microparticles/microvesicles, exosomes, and apoptotic bodies by different productive mechanisms. Clinically, EVs are observed in the blood stream of patients suffering from acute and chronic inflammation evoked by various diseases, and number of EVs in blood flow is often dependent on the inflammatory status and severity of the diseases. To date, it has been reported that small molecules such as RNAs and proteins are encapsulated in EVs; however, the functions of EVs are still unclear in the biological, pathological, and clinical aspects. In this review, we summarize and discuss the biogenesis-based classification, expected function, and pathobiological activities of EVs.

  2. Extracellular vesicles: small bricks for tissue repair/regeneration.

    Science.gov (United States)

    Taverna, Simona; Pucci, Marzia; Alessandro, Riccardo

    2017-02-01

    Extracellular vesicles (EVs) are nano-sized membrane vesicles involved in intercellular communication. EVs have pleiotropic actions in physiological and pathological conditions. The ability of EVs to transports proteins, drugs and nucleic acid, to target specific cells and to increase the stability of therapeutic cargo, make EVs interesting as new devices for the treatment of human disease. In a recently published issue of European journal of pharmaceutical sciences, Silva and colleagues reviewed the ability of EVs to modulate tissue repair and regeneration, focusing on their roles and therapeutic potential as immunomodulatory messengers. In this perspective, we discussed the open questions regarding the dual role of EVs in immune system, as well as the technical limitation of the procedure for EVs isolation and administration in clinical practices. EV-based therapies require further studies to consider EVs as promising candidate for a novel cell-free therapy in the context of regeneration medicine.

  3. Illuminating the physiology of extracellular vesicles

    OpenAIRE

    Choi, Hongyoon; Lee, Dong Soo

    2016-01-01

    Extracellular vesicles play a crucial role in intercellular communication by transmitting biological materials from donor cells to recipient cells. They have pathophysiologic roles in cancer metastasis, neurodegenerative diseases, and inflammation. Extracellular vesicles also show promise as emerging therapeutics, with understanding of their physiology including targeting, distribution, and clearance therefore becoming an important issue. Here, we review recent advances in methods for trackin...

  4. Amyloglucosidase enzymatic reactivity inside lipid vesicles

    Directory of Open Access Journals (Sweden)

    Kim Jin-Woo

    2007-10-01

    Full Text Available Abstract Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG (EC 3.2.1.3 from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC multilamellar vesicles (MLVs and large unilamellar vesicles (LUVs was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.

  5. Extracellular vesicles in coronary artery disease.

    Science.gov (United States)

    Boulanger, Chantal M; Loyer, Xavier; Rautou, Pierre-Emmanuel; Amabile, Nicolas

    2017-05-01

    Membrane vesicles released in the extracellular space are composed of a lipid bilayer enclosing soluble cytosolic material and nuclear components. Extracellular vesicles include apoptotic bodies, exosomes, and microvesicles (also known previously as microparticles). Originating from different subcellular compartments, the role of extracellular vesicles as regulators of transfer of biological information, acting locally and remotely, is now acknowledged. Circulating vesicles released from platelets, erythrocytes, leukocytes, and endothelial cells contain potential valuable biological information for biomarker discovery in primary and secondary prevention of coronary artery disease. Extracellular vesicles also accumulate in human atherosclerotic plaques, where they affect major biological pathways, including inflammation, proliferation, thrombosis, calcification, and vasoactive responses. Extracellular vesicles also recapitulate the beneficial effect of stem cells to treat cardiac consequences of acute myocardial infarction, and now emerge as an attractive alternative to cell therapy, opening new avenues to vectorize biological information to target tissues. Although interest in microvesicles in the cardiovascular field emerged about 2 decades ago, that for extracellular vesicles, in particular exosomes, started to unfold a decade ago, opening new research and therapeutic avenues. This Review summarizes current knowledge on the role of extracellular vesicles in coronary artery disease, and their emerging potential as biomarkers and therapeutic agents.

  6. Synaptic vesicle distribution by conveyor belt.

    Science.gov (United States)

    Moughamian, Armen J; Holzbaur, Erika L F

    2012-03-02

    The equal distribution of synaptic vesicles among synapses along the axon is critical for robust neurotransmission. Wong et al. show that the continuous circulation of synaptic vesicles throughout the axon driven by molecular motors ultimately yields this even distribution. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Urinary extracellular vesicles: biomarkers and beyond

    NARCIS (Netherlands)

    M. Salih (Mahdi)

    2017-01-01

    markdownabstractExtracellular vesicles have been isolated in various body fluids including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly the pathophysiological state of cells lining the nephron. Because

  8. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    Science.gov (United States)

    Wang, Shan Shan H; Held, Richard G; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S

    2016-08-17

    In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near-complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Extracellular vesicles: new players in cardiovascular diseases.

    Science.gov (United States)

    Gaceb, Abderahim; Martinez, Maria Carmen; Andriantsitohaina, Ramaroson

    2014-05-01

    Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. [Transvesical Removal of Seminal Vesicle Cystadenoma].

    Science.gov (United States)

    Takayasu, Kenta; Harada, Jiro; Kawa, Gen; Ota, Syuichi; Sakurai, Takanori

    2015-07-01

    Primary tumors of the seminal vesicles are extremely rare. There have been 25 reports of this tumor from overseas and most cases are cystadenoma. We report a case of seminal vesicle cystadenoma in a 70-year-old man who presented with lower abdominal pain and urinary frequency. A digital rectal examination detected a projecting and hard mass in the right side of the prostate. Magnetic resonance imaging (MRI) showed a 15 cm multiple cystic mass continuous with the right seminal vesicle. A transrectal needle biopsy revealed benign tissue. The tumor was resected using an open transvesical approach that enabled full exposure of the seminal vesicle without damaging the nerves and blood supply of the bladder. Pathology was consistent with a benign seminal vesicle cystadenoma. We describe the natural history, pathology,and surgical approach in this case.

  11. Pushing synaptic vesicles over the RIM.

    Science.gov (United States)

    Kaeser, Pascal S

    2011-05-01

    In a presynaptic nerve terminal, neurotransmitter release is largely restricted to specialized sites called active zones. Active zones consist of a complex protein network, and they organize fusion of synaptic vesicles with the presynaptic plasma membrane in response to action potentials. Rab3-interacting molecules (RIMs) are central components of active zones. In a recent series of experiments, we have systematically dissected the molecular mechanisms by which RIMs operate in synaptic vesicle release. We found that RIMs execute two critical functions of active zones by virtue of independent protein domains. They tether presyanptic Ca(2+) channels to the active zone, and they activate priming of synaptic vesicles by monomerizing homodimeric, constitutively inactive Munc13. These data indicate that RIMs orchestrate synaptic vesicle release into a coherent process. In conjunction with previous studies, they suggest that RIMs form a molecular platform on which plasticity of synaptic vesicle release can operate.

  12. Monosaccharide transport in protein-depleted vesicles from erythrocyte membranes

    National Research Council Canada - National Science Library

    M A Zoccoli; G E Lienhard

    1977-01-01

    .... Based on comparisons between erythrocytes and vesicles with regard to specificity, temparture dependence, and effects of inhibitors, we conclude that sorbose uptake into the vesicles occurs by way...

  13. Applicability of colloid filtration theory in size-distributed, reduced porosity, granular media in the absence of energy barriers.

    Science.gov (United States)

    Pazmino, Eddy F; Ma, Huilian; Johnson, William P

    2011-12-15

    The vast majority of colloid transport experiments use granular porous media with narrow size distribution to facilitate comparison with colloid filtration theory, which represents porous media with a single collector size. In this work we examine retention of colloids ranging in size from 0.21 to 9.1 μm in diameter, in columns packed with uniform and size-distributed borosilicate glass bead porous media with porosity ranging from 0.38 to 0.28. Conditions were favorable to attachment (absent a significant energy barrier). The goal was to determine the applicability of colloid filtration theory to colloid retention in these media. We also directly observed deposition at the pore scale in packed flow cells. The pore domain was characterized via high resolution computerized X-ray micro tomography (HRXMT). The flow field was examined using Lattice-Boltzmann flow simulation methods (LBM). The influence of preferential flow paths on colloid retention in the lowest porosity media was accounted for by correcting the fluid velocity. Straining in pore throats too small to pass was not a significant contributor to colloid retention despite colloid-to-collector size ratios up to 0.05. Mechanistic simulations via the Ma-Pedel-Fife-Johnson correlation equation (MPFJ) for colloid filtration predicted the experimentally observed trends in deposition with porosity when a number-based mean grain size was used.

  14. Modelled Cost-Effectiveness of a Package Size Cap and a Kilojoule Reduction Intervention to Reduce Energy Intake from Sugar-Sweetened Beverages in Australia

    Directory of Open Access Journals (Sweden)

    Michelle Crino

    2017-09-01

    Full Text Available Interventions targeting portion size and energy density of food and beverage products have been identified as a promising approach for obesity prevention. This study modelled the potential cost-effectiveness of: a package size cap on single-serve sugar sweetened beverages (SSBs >375 mL (package size cap, and product reformulation to reduce energy content of packaged SSBs (energy reduction. The cost-effectiveness of each intervention was modelled for the 2010 Australia population using a multi-state life table Markov model with a lifetime time horizon. Long-term health outcomes were modelled from calculated changes in body mass index to their impact on Health-Adjusted Life Years (HALYs. Intervention costs were estimated from a limited societal perspective. Cost and health outcomes were discounted at 3%. Total intervention costs estimated in AUD 2010 were AUD 210 million. Both interventions resulted in reduced mean body weight (package size cap: 0.12 kg; energy reduction: 0.23 kg; and HALYs gained (package size cap: 73,883; energy reduction: 144,621. Cost offsets were estimated at AUD 750.8 million (package size cap and AUD 1.4 billion (energy reduction. Cost-effectiveness analyses showed that both interventions were “dominant”, and likely to result in long term cost savings and health benefits. A package size cap and kJ reduction of SSBs are likely to offer excellent “value for money” as obesity prevention measures in Australia.

  15. AKI Recovery Induced by Mesenchymal Stromal Cell-Derived Extracellular Vesicles Carrying MicroRNAs.

    Science.gov (United States)

    Collino, Federica; Bruno, Stefania; Incarnato, Danny; Dettori, Daniela; Neri, Francesco; Provero, Paolo; Pomatto, Margherita; Oliviero, Salvatore; Tetta, Ciro; Quesenberry, Peter J; Camussi, Giovanni

    2015-10-01

    Phenotypic changes induced by extracellular vesicles have been implicated in mesenchymal stromal cell-promoted recovery of AKI. MicroRNAs are potential candidates for cell reprogramming toward a proregenerative phenotype. The aim of this study was to evaluate whether microRNA deregulation inhibits the regenerative potential of mesenchymal stromal cells and derived extracellular vesicles in a model of glycerol-induced AKI in severe combined immunodeficient mice. We generated mesenchymal stromal cells depleted of Drosha to alter microRNA expression. Drosha-knockdown cells produced extracellular vesicles that did not differ from those of wild-type cells in quantity, surface molecule expression, and internalization within renal tubular epithelial cells. However, these vesicles showed global downregulation of microRNAs. Whereas wild-type mesenchymal stromal cells and derived vesicles administered intravenously induced morphologic and functional recovery in AKI, the Drosha-knockdown counterparts were ineffective. RNA sequencing analysis showed that kidney genes deregulated after injury were restored by treatment with mesenchymal stromal cells and derived vesicles but not with Drosha-knockdown cells and vesicles. Gene ontology analysis showed in AKI an association of downregulated genes with fatty acid metabolism and upregulated genes with inflammation, matrix-receptor interaction, and cell adhesion molecules. These alterations reverted after treatment with wild-type mesenchymal stromal cells and extracellular vesicles but not after treatment with the Drosha-knockdown counterparts. In conclusion, microRNA depletion in mesenchymal stromal cells and extracellular vesicles significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of microRNAs in recovery after AKI. Copyright © 2015 by the American Society of Nephrology.

  16. Structure formation of lipid membranes: Membrane self-assembly and vesicle opening-up to octopus-like micelles

    Science.gov (United States)

    Noguchi, Hiroshi

    2013-02-01

    We briefly review our recent studies on self-assembly and vesicle rupture of lipid membranes using coarse-grained molecular simulations. For single component membranes, lipid molecules self-assemble from random gas states to vesicles via disk-shaped clusters. Clusters aggregate into larger clusters, and subsequently the large disks close into vesicles. The size of vesicles are determined by kinetics than by thermodynamics. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle called bicelle can be formed. When both surfactants have negligibly low critical micelle concentration, it is found that bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and spontaneous curvature of the membrane monolayer.

  17. Modulating the size of ZnO nanorods on SiO2 substrates by incorporating reduced graphene oxide into the seed layer solution

    OpenAIRE

    Tzu-Yi Yu; Min Ruei Wei; Chen Yuan Weng; Wei Ming Su; Chien Cheng Lu; Yu Tzu Chen; Hsiang Chen

    2017-01-01

    In this research, reduced graphene oxide was incorporated into the ZnO seed layer to modulate the rod diameter of ZnO nanorods (NRs) during solgel/hydrothermal growth. To characterize the reduced graphene oxide incorporated ZnO NRs, multiple material analysis techniques including field-emission scanning electron microscopy, surface contact angle measurements, X-ray diffraction, and photoluminescence were used to explore distinct properties of these size modulatable NRs. Results indicate ZnO N...

  18. [Three-dimensional motion analysis for GLUT4 vesicles in TIRF microscopy].

    Science.gov (United States)

    Wu, Xiang-Ping; Li, Jie-Yue; Xu, Ying-Ke; Xu, Ke-Di; Zheng, Xiao-Xiang

    2008-01-01

    In this paper, GLUT4 vesicles are observed in real-time under TIRF microscopy and a new three-dimensional single particle tracking algorithm according to the unique features of TIRF is put forward. Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time and mobile vesicles were segmented by an adaptive background subtraction method. Kalman filtering was then introduced to track the granules so as to reduce the searching range and to avoid the disturbance of background noise and false targets. In the experiments the algorithm was applied in analyzing the long-distance movement of GLUT4 vesicles. The experimental results indicate that the algorithm has achieved robust tracking of the vesicles in the imaging plane and has effectively calculated the position in the direction orthogonal to the imaging plane.

  19. Researches regarding the reducing of burr size by optimising the cutting parameters on a CNC milling machine

    Directory of Open Access Journals (Sweden)

    Biriş Cristina

    2017-01-01

    Full Text Available This paper presents some experimental researches regarding burrs dimensions reduction that appear after the milling process together with an approach to reduce or eliminate the burrs resulted after this process. In order to reduce burrs dimensions, the milling process was executed with different cutting parameters and strategies then the results were evaluated.

  20. Extracellular vesicles as emerging intercellular communicasomes.

    Science.gov (United States)

    Yoon, Yae Jin; Kim, Oh Youn; Gho, Yong Song

    2014-10-01

    All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions.

  1. Kinetic partitioning between aggregation and vesicle permeabilization by modified ADan

    DEFF Research Database (Denmark)

    Nesgaard, Lise W.; Vad, Brian; Christiansen, Gunna

    2009-01-01

    changed to serines to emulate the reduced peptide. SerADan aggregates rapidly at pH 5.0 and 7.5 in a series of conformational transitions to form beta-sheet rich fibril-like structures, which nevertheless do not bind amyloid-specific dyes, probably due to the absence of organized beta-sheet contacts....... Aggregation is prevented at neutral/acidic pH and low ionic strength by anionic lipid vesicles. These vesicles are permeabilized by monomeric SerADan assembling on the membrane to form stable beta-sheet structures which are different from the solution aggregates. In contrast, solution ageing of SerADan first......-fibrillar aggregates can assemble in a series of steps to form a hierarchy of higher-order assemblies, where rapid formation of stable local beta-sheet structure may prevent rearrangement to amyloid proper....

  2. Isolation and characterization of platelet-derived extracellular vesicles.

    Science.gov (United States)

    Aatonen, Maria T; Ohman, Tiina; Nyman, Tuula A; Laitinen, Saara; Grönholm, Mikaela; Siljander, Pia R-M

    2014-01-01

    Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca(2+) ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. The main EV populations were 100-250 nm and over 90% were vesicle subpopulations. Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100-250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

  3. Conformation of charged vesicles: the Debye Huckel and the low curvature limit

    Science.gov (United States)

    Sinha, Kumari Priti; Thaokar, Rochish M., , Prof.

    The shape as well as tension and pressure inside an uncharged vesicle are determined by the reduced volume. These parameters are important for a vesicle or a biological cell, since it can affect bio-physical processes such as osmosis and permeation, interaction with external agents such as bio- macromolecules and thermal fluctuations of the bilayer membrane of a vesicle. Charged membranes are ubiquitous in nature, most biological cell bio-membranes are charged, and therefore the knowledge of shape, tension and pressure of charged vesicles is critical. Additionally, the distribution of charges in the inner and outer leaflets is also important as it can affect the spatial interaction of a bilayer membrane with proteins. This work addresses these issues in the low charge and curvature limit. Our analysis indicates that despite a very strong two-way coupling between the charge and the curvature, the shapes of charged vesicles remain similar to that of uncharged vesicles at comparable reduced volumes, even for reasonable values of total charge. However, the tension and pressure values are higher, and are accurately estimated. Similarly the charge distribution on the outer and inner leaflet is strongly affected by the curvature. The value of spontaneous curvature due to charge redistribution is estimated. The insensitivity of the shape to charges persists even when only the outer leaflet is charged instead of charged inner and outer leaflets

  4. REDUCED-SIZE LIVER-TRANSPLANTATION, SPLIT LIVER-TRANSPLANTATION, AND LIVING-RELATED LIVER-TRANSPLANTATION IN RELATION TO THE DONOR ORGAN SHORTAGE

    NARCIS (Netherlands)

    SLOOFF, MJH

    Because of the shortage of cadaveric donors, three techniques of partial liver grafting have been developed. These techniques are placed in perspective in relation to the organ shortage. Reduced size liver transplantation (RSLTx) is widely used and has results comparable to those from whole liver

  5. The ability of winter grazing to reduce wildfire size, intensity, and fire-induced plant mortality was not demonstrated: A comment on Davies et al. (2015)

    Science.gov (United States)

    A recent study by Davies et al. sought to test whether winter grazing could reduce wildfire size, fire behavior metrics, and fire-induced plant mortality in shrub-grasslands. The authors concluded that ungrazed rangelands may experience more fire-induced mortality of native peren...

  6. Discrete element method modeling of the triboelectric charging of polyethylene particles: Can particle size distribution and segregation reduce the charging?

    Science.gov (United States)

    Konopka, Ladislav; Kosek, Juraj

    2015-10-01

    Polyethylene particles of various sizes are present in industrial gas-dispersion reactors and downstream processing units. The contact of the particles with a device wall as well as the mutual particle collisions cause electrons on the particle surface to redistribute in the system. The undesirable triboelectric charging results in several operational problems and safety risks in industrial systems, for example in the fluidized-bed polymerization reactor. We studied the charging of polyethylene particles caused by the particle-particle interactions in gas. Our model employs the Discrete Element Method (DEM) describing the particle dynamics and incorporates the ‘Trapped Electron Approach’ as the physical basis for the considered charging mechanism. The model predicts the particle charge distribution for systems with various particle size distributions and various level of segregation. Simulation results are in a qualitative agreement with experimental observations of similar particulate systems specifically in two aspects: 1) Big particles tend to gain positive charge and small particles the negative one. 2) The wider the particle size distribution is, the more pronounced is the charging process. Our results suggest that not only the size distribution, but also the effect of the spatial segregation of the polyethylene particles significantly influence the resulting charge distribution ‘generated’ in the system. The level of particle segregation as well as the particle size distribution of polyethylene particles can be in practice adjusted by the choice of supported catalysts, by the conditions in the fluidized-bed polymerization reactor and by the fluid dynamics. We also attempt to predict how the reactor temperature affects the triboelectric charging of particles.

  7. Two to five repeated measurements per patient reduced the required sample size considerably in a randomized clinical trial for patients with inflammatory rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Smedslund Geir

    2013-02-01

    Full Text Available Abstract Background Patient reported outcomes are accepted as important outcome measures in rheumatology. The fluctuating symptoms in patients with rheumatic diseases have serious implications for sample size in clinical trials. We estimated the effects of measuring the outcome 1-5 times on the sample size required in a two-armed trial. Findings In a randomized controlled trial that evaluated the effects of a mindfulness-based group intervention for patients with inflammatory arthritis (n=71, the outcome variables Numerical Rating Scales (NRS (pain, fatigue, disease activity, self-care ability, and emotional wellbeing and General Health Questionnaire (GHQ-20 were measured five times before and after the intervention. For each variable we calculated the necessary sample sizes for obtaining 80% power (α=.05 for one up to five measurements. Two, three, and four measures reduced the required sample sizes by 15%, 21%, and 24%, respectively. With three (and five measures, the required sample size per group was reduced from 56 to 39 (32 for the GHQ-20, from 71 to 60 (55 for pain, 96 to 71 (73 for fatigue, 57 to 51 (48 for disease activity, 59 to 44 (45 for self-care, and 47 to 37 (33 for emotional wellbeing. Conclusions Measuring the outcomes five times rather than once reduced the necessary sample size by an average of 27%. When planning a study, researchers should carefully compare the advantages and disadvantages of increasing sample size versus employing three to five repeated measurements in order to obtain the required statistical power.

  8. Illuminating the physiology of extracellular vesicles.

    Science.gov (United States)

    Choi, Hongyoon; Lee, Dong Soo

    2016-04-16

    Extracellular vesicles play a crucial role in intercellular communication by transmitting biological materials from donor cells to recipient cells. They have pathophysiologic roles in cancer metastasis, neurodegenerative diseases, and inflammation. Extracellular vesicles also show promise as emerging therapeutics, with understanding of their physiology including targeting, distribution, and clearance therefore becoming an important issue. Here, we review recent advances in methods for tracking and imaging extracellular vesicles in vivo and critically discuss their systemic distribution, targeting, and kinetics based on up-to-date evidence in the literature.

  9. Synaptopathy under conditions of altered gravity: changes in synaptic vesicle fusion and glutamate release.

    Science.gov (United States)

    Krisanova, N V; Trikash, I O; Borisova, T A

    2009-12-01

    Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO ( approximately 10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[(14)C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca(2+) or Mg(2+)/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.

  10. Glioblastoma extracellular vesicles: reservoirs of potential biomarkers

    Directory of Open Access Journals (Sweden)

    Redzic JS

    2014-02-01

    Full Text Available Jasmina S Redzic,1 Timothy H Ung,2 Michael W Graner2 1Skaggs School of Pharmacy and Pharmaceutical Sciences, 2Department of Neurosurgery, School of Medicine, University of Colorado Denver, Aurora, CO, USA Abstract: Glioblastoma multiforme (GBM is the most frequent and most devastating of the primary central nervous system tumors, with few patients living beyond 2 years postdiagnosis. The damage caused by the disease and our treatments for the patients often leave them physically and cognitively debilitated. Generally, GBMs appear after very short clinical histories and are discovered by imaging (using magnetic resonance imaging [MRI], and the diagnosis is validated by pathology, following surgical resection. The treatment response and diagnosis of tumor recurrence are also tracked by MRI, but there are numerous problems encountered with these monitoring modalities, such as ambiguous interpretation and forms of pseudoprogression. Diagnostic, prognostic, and predictive biomarkers would be an immense boon in following treatment schemes and in determining recurrence, which often requires an invasive intracranial biopsy to verify imaging data. Extracellular vesicles (EVs are stable, membrane-enclosed, virus-sized particles released from either the cell surface or from endosomal pathways that lead to the systemic release of EVs into accessible biofluids, such as serum/plasma, urine, cerebrospinal fluid, and saliva. EVs carry a wide variety of proteins, nucleic acids, lipids, and other metabolites, with many common features but with enough individuality to be able to identify the cell of origin of the vesicles. These components, if properly interrogated, could allow for the identification of tumor-derived EVs in biofluids, indicating tumor progression, relapse, or treatment failure. That knowledge would allow clinicians to continue with treatment regimens that were actually effective or to change course if the therapies were failing. Here, we review

  11. Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.

    Science.gov (United States)

    Athman, Jaffre J; Wang, Ying; McDonald, David J; Boom, W Henry; Harding, Clifford V; Wearsch, Pamela A

    2015-08-01

    Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected

  12. Dendritic nonlinearities reduce network size requirements and mediate ON and OFF states of persistent activity in a PFC microcircuit model.

    Directory of Open Access Journals (Sweden)

    Athanasia Papoutsi

    2014-07-01

    Full Text Available Technological advances have unraveled the existence of small clusters of co-active neurons in the neocortex. The functional implications of these microcircuits are in large part unexplored. Using a heavily constrained biophysical model of a L5 PFC microcircuit, we recently showed that these structures act as tunable modules of persistent activity, the cellular correlate of working memory. Here, we investigate the mechanisms that underlie persistent activity emergence (ON and termination (OFF and search for the minimum network size required for expressing these states within physiological regimes. We show that (a NMDA-mediated dendritic spikes gate the induction of persistent firing in the microcircuit. (b The minimum network size required for persistent activity induction is inversely proportional to the synaptic drive of each excitatory neuron. (c Relaxation of connectivity and synaptic delay constraints eliminates the gating effect of NMDA spikes, albeit at a cost of much larger networks. (d Persistent activity termination by increased inhibition depends on the strength of the synaptic input and is negatively modulated by dADP. (e Slow synaptic mechanisms and network activity contain predictive information regarding the ability of a given stimulus to turn ON and/or OFF persistent firing in the microcircuit model. Overall, this study zooms out from dendrites to cell assemblies and suggests a tight interaction between dendritic non-linearities and network properties (size/connectivity that may facilitate the short-memory function of the PFC.

  13. Classification, Functions, and Clinical Relevance of Extracellular Vesicles

    NARCIS (Netherlands)

    van der Pol, Edwin; Böing, Anita N.; Harrison, Paul; Sturk, Augueste; Nieuwland, Rienk

    2012-01-01

    Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the

  14. Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer

    OpenAIRE

    Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M.; Carracedo, Arkaitz

    2016-01-01

    Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data in...

  15. Electroformation of Giant Unilamellar Vesicles from Native Membranes and Organic Lipid Mixtures for the Study of Lipid Domains under Physiological Ionic-Strength Conditions

    DEFF Research Database (Denmark)

    Montes, Ruth; Ahyayauch, Hasna; Ibarguren, Maitane

    2010-01-01

    Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allows direct visualization of particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols for ...

  16. Development and characterization of nanopore system for nano-vesicle analysis

    Science.gov (United States)

    Goyal, Gaurav

    -translocational deformation behavior; however, they appear to be less affected by the deforming force inside the nanopore compared to the DOPC liposomes. We believe, the results of this research will bring about a novel nano-bioanalytical platform that can be used to capture comprehensive size and deformability data on nano-vesicles with high temporal resolution.

  17. Modulating the size of ZnO nanorods on SiO2 substrates by incorporating reduced graphene oxide into the seed layer solution

    Directory of Open Access Journals (Sweden)

    Tzu-Yi Yu

    2017-06-01

    Full Text Available In this research, reduced graphene oxide was incorporated into the ZnO seed layer to modulate the rod diameter of ZnO nanorods (NRs during solgel/hydrothermal growth. To characterize the reduced graphene oxide incorporated ZnO NRs, multiple material analysis techniques including field-emission scanning electron microscopy, surface contact angle measurements, X-ray diffraction, and photoluminescence were used to explore distinct properties of these size modulatable NRs. Results indicate ZnO NRs with smaller diameters could be observed with more reduced graphene oxide added into the ZnO seed layer. Furthermore, better crystallinity, higher hydrophobicity and lower defect concentration could be obtained with more amount of reduced graphene oxide added into the ZnO seed layer. The modulatable reduced graphene oxide-incorporated ZnO NRs growth is promising for future ZnO NRs based nanodevice applications.

  18. Modulating the size of ZnO nanorods on SiO2 substrates by incorporating reduced graphene oxide into the seed layer solution

    Science.gov (United States)

    Yu, Tzu-Yi; Wei, Min Ruei; Weng, Chen Yuan; Su, Wei Ming; Lu, Chien Cheng; Chen, Yu Tzu; Chen, Hsiang

    2017-06-01

    In this research, reduced graphene oxide was incorporated into the ZnO seed layer to modulate the rod diameter of ZnO nanorods (NRs) during solgel/hydrothermal growth. To characterize the reduced graphene oxide incorporated ZnO NRs, multiple material analysis techniques including field-emission scanning electron microscopy, surface contact angle measurements, X-ray diffraction, and photoluminescence were used to explore distinct properties of these size modulatable NRs. Results indicate ZnO NRs with smaller diameters could be observed with more reduced graphene oxide added into the ZnO seed layer. Furthermore, better crystallinity, higher hydrophobicity and lower defect concentration could be obtained with more amount of reduced graphene oxide added into the ZnO seed layer. The modulatable reduced graphene oxide-incorporated ZnO NRs growth is promising for future ZnO NRs based nanodevice applications.

  19. Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

    DEFF Research Database (Denmark)

    Vind-Kezunovic, D.; Nielsen, C.H.; Wojewodzka, U.

    2008-01-01

    Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L(o)) and liquid-disordered (L(d)) phases. Here, we show mum-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the ...... mum-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events Udgivelsesdato: 2008/11....... Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total 'L(o)/L(d)' boundary line tension arising from the coalescence of smaller L(d)-like domains makes it energetically favourable for L(d)-like domains to bend from flat...

  20. Circumferential bone grafting around an absorbable gelatin sponge core reduced the amount of grafted bone in the induced membrane technique for critical-size defects of long bones.

    Science.gov (United States)

    Cho, Jae-Woo; Kim, Jinil; Cho, Won-Tae; Kim, Jin-Kak; Song, Jong Hoon; Kim, Hyung-Jin; Masquelet, Alain C; Oh, Jong-Keon

    2017-10-01

    The objectives of the study were to introduce a circumferential bone graft around an absorbable gelatin sponge core using an induced membrane technique, to assess its ability to reduce the required amount of graft and to maintain the bone graft, and to evaluate the clinical outcomes in the management of critical-size bone defects. Circumferential bone grafting using a staged induced membrane technique for managing critical-size bone defects was performed in 21 patients. Postoperative computed tomography scans were performed 7days after Hemovac drain removal and 3 months after bone grafting. Volumetric measurements of the defect size, gelatin sponge proportion, and amount of grafted bone were performed by two independent observers using three-dimensional (3D) software. The critical-size defects were located at the metadiaphyseal area of 11 tibias, eight femurs, and two humeri. The average defect size was 8.9cm in length and 65.2cm3 in volume. The absorbable gelatin sponge core replaced 21.4% (average) of the defect volume. There was no significant deterioration in the shape of the grafted bone among the serial 3D models. Eighteen patients (86%) were healed radiographically at 9.1 months (average). Our study suggests that circumferential bone grafting in association with the induced membrane technique could reduce the required amount of bone graft and adequately maintain graft position and shape, with favourable clinical outcomes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Hybrid, Nanoscale Phospholipid/Block Copolymer Vesicles

    Directory of Open Access Journals (Sweden)

    Bo Liedberg

    2013-09-01

    Full Text Available Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chemical versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by preparing giant unilamellar vesicles (GUVs via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diameter of the same composition prepared by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide (PB-PEO/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and molecular imaging/sensing applications and clearly open up new avenues for further investigation.

  2. Stability of Spherical Vesicles in Electric Fields

    Science.gov (United States)

    2010-01-01

    The stability of spherical vesicles in alternating (ac) electric fields is studied theoretically for asymmetric conductivity conditions across their membranes. The vesicle deformation is obtained from a balance between the curvature elastic energies and the work done by the Maxwell stresses. The present theory describes and clarifies the mechanisms for the four types of morphological transitions observed experimentally on vesicles exposed to ac fields in the frequency range from 500 to 2 × 107 Hz. The displacement currents across the membranes redirect the electric fields toward the membrane normal to accumulate electric charges by the Maxwell−Wagner mechanism. These accumulated electric charges provide the underlying molecular mechanism for the morphological transitions of vesicles as observed on the micrometer scale. PMID:20575588

  3. Kinetic regulation of coated vesicle secretion

    CERN Document Server

    Foret, Lionel

    2008-01-01

    The secretion of vesicles for intracellular transport often rely on the aggregation of specialized membrane-bound proteins into a coat able to curve cell membranes. The nucleation and growth of a protein coat is a kinetic process that competes with the energy-consuming turnover of coat components between the membrane and the cytosol. We propose a generic kinetic description of coat assembly and the formation of coated vesicles, and discuss its implication to the dynamics of COP vesicles that traffic within the Golgi and with the Endoplasmic Reticulum. We show that stationary coats of fixed area emerge from the competition between coat growth and the recycling of coat components, in a fashion resembling the treadmilling of cytoskeletal filaments. We further show that the turnover of coat components allows for a highly sensitive switching mechanism between a quiescent and a vesicle producing membrane, upon a slowing down of the exchange kinetics. We claim that the existence of this switching behaviour, also tri...

  4. Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis

    Directory of Open Access Journals (Sweden)

    Stephanie N. Hurwitz

    2016-07-01

    Full Text Available Background: Extracellular vesicles (EVs are important mediators of cell-to-cell communication in healthy and pathological environments. Because EVs are present in a variety of biological fluids and contain molecular signatures of their cell or tissue of origin, they have great diagnostic and prognostic value. The ability of EVs to deliver biologically active proteins, RNAs and lipids to cells has generated interest in developing novel therapeutics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis and secretion remain unknown. Methods: Here, we characterized vesicle secretion across the NCI-60 panel of human cancer cells by nanoparticle tracking analysis. Using CellMiner, the quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. Results: Gene products positively associated with the quantity of exosomal-sized vesicles included vesicular trafficking classes of proteins with Rab GTPase function and sphingolipid metabolism. Positive correlates of larger microvesicle-sized vesicle secretion included gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. Conclusion: These observations reveal new insights into genes involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. This study offers a foundation for further exploration of targets involved in EV biogenesis and secretion.

  5. On the growth of walled cells: From shells to vesicles.

    Science.gov (United States)

    Boudaoud, Arezki

    2003-03-01

    The growth of isolated walled cells is investigated. Examples of such cells range from bacteria to giant algae, and include cochlear hair, plant root hair, fungi and yeast cells. They are modeled as elastic shells inflated by a liquid. Cell growth is driven by fluid pressure and is similar to a plastic deformation of the wall. The requirement of mechanical equilibrium leads to two new scaling laws for cell size that are in quantitative agreement with the compiled biological data. Given these results, possible shapes for growing cells are computed by analogy with those of vesicle membranes.

  6. Growth of Walled Cells: From Shells to Vesicles

    Science.gov (United States)

    Boudaoud, Arezki

    2003-07-01

    The growth of isolated walled cells is investigated. Examples of such cells range from bacteria to giant algae, and include cochlear hair, plant root hair, fungi, and yeast cells. They are modeled as elastic shells containing a liquid. Cell growth is driven by fluid pressure and is is similar to a plastic deformation of the wall. The requirement of mechanical equilibrium leads to two new scaling laws for cell size that are in quantitative agreement with the compiled biological data. Given these results, possible shapes for growing cells are computed by analogy with those of vesicle membranes.

  7. Mutations in Synaptojanin Disrupt Synaptic Vesicle Recycling

    OpenAIRE

    Harris, Todd W.; Hartwieg, Erika; Horvitz, H. Robert; Jorgensen, Erik M.

    2000-01-01

    Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in ...

  8. Concentration-Independent Spontaneously Forming Biomimetric Vesicles

    Science.gov (United States)

    Nieh, M.-P.; Harroun, T. A.; Raghunathan, V. A.; Glinka, C. J.; Katsaras, J.

    2003-10-01

    In this Letter we present small-angle neutron scattering data from a biomimetic system composed of the phospholipids dimyristoyl and dihexanoyl phosphorylcholine (DMPC and DHPC, respectively). Doping DMPC-DHPC multilamellar vesicles with either the negatively charged lipid dimyristoyl phosphorylglycerol (DMPG, net charge -1) or the divalent cation, calcium (Ca2+), leads to the spontaneous formation of energetically stabilized monodisperse unilamellar vesicles whose radii are concentration independent and in contrast with previous experimental observations.

  9. Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

    OpenAIRE

    Aizea Morales-Kastresana; Bill Telford; Musich, Thomas A.; Katherine McKinnon; Cassandra Clayborne; Zach Braig; Ari Rosner; Thorsten Demberg; Watson, Dionysios C.; Karpova, Tatiana S.; Freeman, Gordon J.; DeKruyff, Rosemarie H.; Pavlakis, George N.; Masaki Terabe; Marjorie Robert-Guroff

    2017-01-01

    Extracellular vesicles (EVs), including exosomes and microvesicles, are 30?800?nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be brig...

  10. Early transient presence of implanted bone marrow stem cells reduces lesion size after cerebral ischaemia in adult rats

    NARCIS (Netherlands)

    Keimpema, E.; Fokkens, M. R.; Nagy, Z.; Agoston, V.; Luiten, P. G. M.; Nyakas, C.; Boddeke, H. W. G. M.; Copray, J. C. V. M.

    Aims: Previous studies on the therapeutic time window for intravascular administration of bone marrow stem cells (BMSCs) after stroke have shown that early intervention (from 3 h after onset) in the middle cerebral artery occlusion (MCAO) rat model is the most effective approach to reduce ischaemic

  11. Cellular Phenotype and Extracellular Vesicles: Basic and Clinical Considerations

    OpenAIRE

    Quesenberry, Peter J.; Goldberg, Laura R.; Aliotta, Jason M.; Mark S Dooner; Pereira, Mandy G.; Wen, Sicheng; Camussi, Giovanni

    2014-01-01

    Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently, vesicles were studied as possible vaccines and, most recently, there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either the lung o...

  12. Hierarchical unilamellar vesicles of controlled compositional heterogeneity.

    Directory of Open Access Journals (Sweden)

    Maik Hadorn

    Full Text Available Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors.

  13. Endothelial Extracellular Vesicles-Promises and Challenges.

    Science.gov (United States)

    Hromada, Carina; Mühleder, Severin; Grillari, Johannes; Redl, Heinz; Holnthoner, Wolfgang

    2017-01-01

    Extracellular vesicles, including exosomes, microparticles, and apoptotic bodies, are phospholipid bilayer-enclosed vesicles that have once been considered as cell debris lacking biological functions. However, they have recently gained immense interest in the scientific community due to their role in intercellular communication, immunity, tissue regeneration as well as in the onset, and progression of various pathologic conditions. Extracellular vesicles of endothelial origin have been found to play a versatile role in the human body, since they are on the one hand known to contribute to cardiovascular diseases, but on the other hand have also been reported to promote endothelial cell survival. Hence, endothelial extracellular vesicles hold promising therapeutic potential to be used as a new tool to detect as well as treat a great number of diseases. This calls for clinically approved, standardized, and efficient isolation and characterization protocols to harvest and purify endothelial extracellular vesicles. However, such methods and techniques to fulfill stringent requirements for clinical trials have yet to be developed or are not harmonized internationally. In this review, recent advances and challenges in the field of endothelial extracellular vesicle research are discussed and current problems and limitations regarding isolation and characterization are pointed out.

  14. Amyloid precursor protein knockout diminishes synaptic vesicle proteins at the presynaptic active zone in mouse brain.

    Science.gov (United States)

    Laßek, Melanie; Weingarten, Jens; Acker-Palmer, Amparo; Bajjalieh, Sandra M; Muller, Ulrike; Volknandt, Walter

    2014-01-01

    The amyloid precursor protein (APP) has previously been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a presynaptic active zone-localized pool. By analyzing homozygous APP knockout mice we evaluated the impact of APP on synaptic vesicle protein abundance at synaptic release sites. Following immunopurification of synaptic vesicles and the attached presynaptic plasma membrane, individual proteins were subjected to quantitative Western blot analysis. We demonstrate that APP deletion in knockout animals reduces the abundance of the synaptic vesicle proteins synaptophysin, synaptotagmin-1, and SV2A at the presynaptic active zone. Conversely, deletion of the additional APP family members, APLP1 and APLP2 resulted in an increase in synaptophysin, synaptogamin-1, and SV2A abundance. When transmembrane APP is lacking in APPsα-KI/APLP2-KO mice synaptic vesicle protein abundance corresponds to that in APP -KO mice. Deletion of the synaptic vesicle protein 2 (SV2) A and B had no effect on APP and synaptophysin abundance but decreased synaptotagmin-1. Our data suggest that APP controls the abundance of synaptic vesicle proteins at the presynaptic release sites and thus impacts synaptic transmission.

  15. A Vesicle-to-Worm Transition Provides a New High-Temperature Oil Thickening Mechanism.

    Science.gov (United States)

    Derry, Matthew J; Mykhaylyk, Oleksandr O; Armes, Steven P

    2017-02-06

    Diblock copolymer vesicles are prepared via RAFT dispersion polymerization directly in mineral oil. Such vesicles undergo a vesicle-to-worm transition on heating to 150 °C, as judged by TEM and SAXS. Variable-temperature 1 H NMR spectroscopy indicates that this transition is the result of surface plasticization of the membrane-forming block by hot solvent, effectively increasing the volume fraction of the stabilizer block and so reducing the packing parameter for the copolymer chains. The rheological behavior of a 10 % w/w copolymer dispersion in mineral oil is strongly temperature-dependent: the storage modulus increases by five orders of magnitude on heating above the critical gelation temperature of 135 °C, as the non-interacting vesicles are converted into weakly interacting worms. SAXS studies indicate that, on average, three worms are formed per vesicle. Such vesicle-to-worm transitions offer an interesting new mechanism for the high-temperature thickening of oils. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Sites of glucose transporter-4 vesicle fusion with the plasma membrane correlate spatially with microtubules.

    Directory of Open Access Journals (Sweden)

    Jennine M Dawicki-McKenna

    Full Text Available In adipocytes, vesicles containing glucose transporter-4 (GLUT4 redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin, preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.

  17. Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules

    Science.gov (United States)

    Dawicki-McKenna, Jennine M.; Goldman, Yale E.; Ostap, E. Michael

    2012-01-01

    In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion. PMID:22916292

  18. Syntaxin 1B, but not syntaxin 1A, is necessary for the regulation of synaptic vesicle exocytosis and of the readily releasable pool at central synapses.

    Directory of Open Access Journals (Sweden)

    Tatsuya Mishima

    Full Text Available Two syntaxin 1 (STX1 isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

  19. Self-assembly in aqueous solution of wheel-shaped Mo154 oxide clusters into vesicles.

    Science.gov (United States)

    Liu, Tianbo; Diemann, Ekkehard; Li, Huilin; Dress, Andreas W M; Müller, Achim

    2003-11-06

    Surfactants and membrane lipids readily assemble into complex structures such as micelles, liposomes or hollow vesicles owing to their amphiphilic character-the fact that part of their structure is attracted to polar environments while another part is attracted to non-polar environments. The self-assembly of complex structures also occurs in polyoxometallate chemistry, as exemplified by the molybdenum blue solutions known for centuries. But while the presence of nanometre-sized metal oxide aggregates in these solutions has long been recognized, unravelling the composition and formation process of these aggregates proved difficult. Recent work has indicated that discrete, wheel-shaped mixed-valence polyoxomolybdate clusters of the type [Mo154] (refs 2-4) assemble into well-defined nanometre-sized aggregates, including spherical structures. Here we report light-scattering data and transmission electron microscopy images of hollow spherical structures with an average, almost monodisperse radius of about 45 nm and composed of approximately 1,165 [Mo154] wheel-shaped clusters. The clusters appear to lie flat and homogeneously distributed on the vesicle surface. Unlike conventional lipid vesicles, the structures we observe are not stabilized by hydrophobic interactions. Instead, we believe the polyoxomolybdate-based vesicles form owing to a subtle interplay between short-range van der Waals attraction and long-range electrostatic repulsion, with important further stabilization arising from hydrogen bonding involving water molecules encapsulated between the wheel-shaped clusters and in the vesicles' interior.

  20. Extracellular membrane vesicles in blood products-biology and clinical relevance

    Directory of Open Access Journals (Sweden)

    Emilija Krstova Krajnc

    2016-01-01

    Full Text Available Extracellular membrane vesicles are fragments shed from plasma membranes off all cell types that are undergoing apoptosis or are being subjected to various types of stimulation or stress.  Even in the process of programmed cell death (apoptosis, cell fall apart of varying size vesicles. They expose phosphatidylserine (PS on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cellular origin. Extracellular membrane vesicles have been isolated from many types of biological fluids, including serum, cerebrospinal fluid, urine, saliva, tears and conditioned culture medium. Flow cytometry is one of the many different methodological approaches that have been used to analyze EMVs. The method attempts to characterize the EMVs cellular origin, size, population, number, and structure. EMVs are present and accumulate in blood products (erythrocytes, platelets as well as in fresh frozen plasma during storage. The aim of this review is to highlight the importance of extracellular vesicles as a cell-to-cell communication system and the role in the pathogenesis of different diseases. Special emphasis will be given to the implication of extracellular membrane vesicles in blood products and their clinical relevance. Although our understanding of the role of  EMVs in disease is far from comprehensive, they display promise as biomarkers for different diseases in the future and also as a marker of quality and safety in the quality control of blood products.

  1. α-Synuclein Dimers Impair Vesicle Fission during Clathrin-Mediated Synaptic Vesicle Recycling

    Directory of Open Access Journals (Sweden)

    Audrey T. Medeiros

    2017-12-01

    Full Text Available α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV trafficking. In Parkinson’s disease (PD and several other neurodegenerative disorders, aberrant oligomerization and aggregation of α-synuclein lead to synaptic dysfunction and neurotoxicity. Despite evidence that α-synuclein oligomers are generated within neurons under physiological conditions, and that altering the balance of monomers and oligomers contributes to disease pathogenesis, how each molecular species of α-synuclein impacts SV trafficking is currently unknown. To address this, we have taken advantage of lamprey giant reticulospinal (RS synapses, which are accessible to acute perturbations via axonal microinjection of recombinant proteins. We previously reported that acute introduction of monomeric α-synuclein inhibited SV recycling, including effects on the clathrin pathway. Here, we report the effects of α-synuclein dimers at synapses. Similar to monomeric α-synuclein, both recombinant α-synuclein dimers that were evaluated bound to small liposomes containing anionic lipids in vitro, but with reduced efficacy. When introduced to synapses, the α-synuclein dimers also induced SV recycling defects, which included a build up of clathrin-coated pits (CCPs with constricted necks that were still attached to the plasma membrane, a phenotype indicative of a vesicle fission defect. Interestingly, both α-synuclein dimers induced longer necks on CCPs as well as complex, branching membrane tubules, which were distinct from the CCPs induced by a dynamin inhibitor, Dynasore. In contrast, monomeric α-synuclein induced a buildup of free clathrin-coated vesicles (CCVs, indicating an inhibition of clathrin-mediated endocytosis at a later stage during the clathrin uncoating process. Taken together, these data further support the conclusion that excess α-synuclein impairs SV recycling. The data additionally reveal that monomeric and dimeric α-synuclein produce

  2. Light-induced DELTApH and DELTApsi in halobacterial vesicles related to sodium transport

    Energy Technology Data Exchange (ETDEWEB)

    Kamo, N.; Racanclli, T.; Packer, L.

    1986-01-01

    Membranes of Halobacterium halobium contain two retinoproteins, baceteriorhodopsin (BR/sub 568nm/) and halorhodopsin (HR/sub 588nm/). We have investigated the light- and sodium-dependent activities in vesicles from the HR containing R/sub 1/mR strain, and the BR + HR containing S/sub 9/ strain to study energy conversion and ion flow mechanisms. Simultaneous ..delta..pH and ..delta..psi measurements have been made with electrodes. In R/sub 1/mR vesicles, -..delta..psi and H/sup +/ uptake occurs in NaCl but not in KCl medium. In S/sub 9/ vesicles, net H/sup +/ extrusion is reduced at high light intensity in NaCl but not KCl medium. Such results indicate Na/sup +//H/sup +/ exchange in vesicles from both strains. As S/sub 9/ contains BR + HR, it is unclear whether the Na/sup +/ extrusion is due to a Na/sup +//H/sup +/ antiporter and/or HR which has been proposed to be a light driven Na/sup +/ pump. To evaluate these concepts for Na/sup +/ transport, the light intensity dependence and action of several membrane transport active agents have been compared. Digitoxin, electro-neutral exchangers (triphenyltin and monensin), and phloretin yielded similar results for HR (R/sub 1/mR) and HR + BR (S/sub 9/) vesicles. Moreover treatment of vesicles with carboxyl reacting reagents inhibited Na/sup +/ dependent activity in both types of vesicles. Thus, common mechanisms of Na/sup +/ transport are indicated in S/sub 9/ and R/sub 1/mR vesicles. 22 refs., 9 figs., 1 tab.

  3. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes.

    Science.gov (United States)

    Hashimoto, Takeshi; Yokokawa, Takumi; Endo, Yuriko; Iwanaka, Nobumasa; Higashida, Kazuhiko; Taguchi, Sadayoshi

    2013-10-11

    A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Differentiated 3T3-L1 adipocytes were incubated at 5% O2 for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (psize via decreased glucose uptake and lipogenic protein expression and increased basal lipolysis. Such an hypoxia-induced decrease in lipogenesis may be an attractive therapeutic target against lipid-associated metabolic diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. The Use of Active Elements to Reduce the Size and Weight of Passive Components in Adjustable Speed Drives

    DEFF Research Database (Denmark)

    Maheshwari, Ram Krishan

    . To facilitate the design and analysis of the active damping terms, the effects of the active damping terms on the machine current and the DC-link voltage of the drive system are discussed. A design recommendation for the proposed active damping terms is given. Simulation and experimental results verifying......Adjustable speed drives are considered as workhorse of industry due to various applications in different kind of industries. According to a market survey, low voltage drives are most profitable in the drive industries. The drive consists of a machine and a converter, which processes grid power...... and converts into usable power for the machine. The converter consists of active and passive components. The size, weight, and volume of the drive are decided by the passive components since they typically contribute to 75% of the weight and volume of the drive. Most popular topology for the low voltage drive...

  5. Sizing community energy storage systems to reduce transformer overloading with emphasis on plug-in electric vehicle loads

    Science.gov (United States)

    Trowler, Derik Wesley

    The research objective of this study was to develop a sizing method for community energy storage systems with emphasis on preventing distribution transformer overloading due to plug-in electric vehicle charging. The method as developed showed the formulation of a diversified load profile based upon residential load data for several customers on the American Electric Power system. Once a load profile was obtained, plug-in electric vehicle charging scenarios which were based upon expected adoption and charging trends were superimposed on the load profile to show situations where transformers (in particular 25 kVA, 50 kVA, and 100 kVA) would be overloaded during peak hours. Once the total load profiles were derived, the energy and power requirements of community energy storage systems were calculated for a number of scenarios with different combinations of numbers of homes and plug-in electric vehicles. The results were recorded and illustrated into charts so that one could determine the minimum size per application. Other topics that were covered in this thesis were the state of the art and future trends in plug-in electric vehicle and battery chemistry adoption and development. The goal of the literature review was to confirm the already suspected notion that Li-ion batteries are best suited and soon to be most cost-effective solution for applications requiring small, efficient, reliable, and light-weight battery systems such as plug-in electric vehicles and community energy storage systems. This thesis also includes a chapter showing system modeling in MATLAB/SimulinkRTM. All in all, this thesis covers a wide variety of considerations involved in the designing and deploying of community energy storage systems intended to mitigate the effects of distribution transformer overloading.

  6. Lipid lateral organization on giant unilamellar vesicles containing lipopolysaccharides

    DEFF Research Database (Denmark)

    Kubiak, Jakub; Brewer, Jonathan R.; Hansen, Søren

    2011-01-01

    -Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles...... the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains......, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to ∼15 mol %). However, fluorescence correlation...

  7. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation

    Science.gov (United States)

    Bobrie, Angélique; Colombo, Marina; Krumeich, Sophie; Raposo, Graça; Théry, Clotilde

    2012-01-01

    Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles. PMID:24009879

  8. Single phase bi-directional AC-DC converter with reduced passive components size and common mode electro-magnetic interference

    Energy Technology Data Exchange (ETDEWEB)

    Mi, Chris; Li, Siqi

    2017-01-31

    A bidirectional AC-DC converter is presented with reduced passive component size and common mode electro-magnetic interference. The converter includes an improved input stage formed by two coupled differential inductors, two coupled common and differential inductors, one differential capacitor and two common mode capacitors. With this input structure, the volume, weight and cost of the input stage can be reduced greatly. Additionally, the input current ripple and common mode electro-magnetic interference can be greatly attenuated, so lower switching frequency can be adopted to achieve higher efficiency.

  9. The Cladophora glomerata Enriched by Biosorption Process in Cr(III Improves Viability, and Reduces Oxidative Stress and Apoptosis in Equine Metabolic Syndrome Derived Adipose Mesenchymal Stromal Stem Cells (ASCs and Their Extracellular Vesicles (MV’s

    Directory of Open Access Journals (Sweden)

    Krzysztof Marycz

    2017-12-01

    Full Text Available This study investigated in vitro effects of freshwater alga Cladophora glomerata water extract enriched during a biosorption process in Cr(III trivalent chromium and chromium picolinate on adipose-derived mesenchymal stromal stem cells (ASCs and extracellular microvesicles (MVs in equine metabolic syndrome-affected horses. Chemical characterisation of natural Cladophora glomerata was performed with special emphasis on: vitamin C, vitamin E, total phenols, fatty acids, free and protein-bound amino acids as well as measured Cr in algal biomass. To examine the influence of Cladophora glomerata water extracts, in vitro viability, oxidative stress factor accumulation, apoptosis, inflammatory response, biogenesis of mitochondria, autophagy in ASCs of EMS and secretory activity manifested by MV release were investigated. For this purpose, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, TEM, FIB observations, mRNA and microRNA expression by RT-qPCR were applied. The extract of Cladophora glomerata enriched with Cr(III ions reduced apoptosis and inflammation in ASCs of EMS horses through improvement of mitochondrial dynamics, decreasing of PDK4 expression and reduction of endoplastic reticulum stress. Moreover, it was found, that Cladophora glomerata and Cr(III induce antioxidative protection coming from enhanced SOD activity Therefore, Cladophora glomerata enriched with Cr(III ions might become an interesting future therapeutic agent in the pharmacological treatment of EMS horses.

  10. Extracellular Vesicles in Metabolic Syndrome.

    Science.gov (United States)

    Martínez, M Carmen; Andriantsitohaina, Ramaroson

    2017-05-12

    Metabolic syndrome defines a cluster of interrelated risk factors for cardiovascular disease and diabetes mellitus. These factors include metabolic abnormalities, such as hyperglycemia, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, high blood pressure, and obesity, mainly central adiposity. In this context, extracellular vesicles (EVs) may represent novel effectors that might help to elucidate disease-specific pathways in metabolic disease. Indeed, EVs (a terminology that encompasses microparticles, exosomes, and apoptotic bodies) are emerging as a novel mean of cell-to-cell communication in physiology and pathology because they represent a new way to convey fundamental information between cells. These microstructures contain proteins, lipids, and genetic information able to modify the phenotype and function of the target cells. EVs carry specific markers of the cell of origin that make possible monitoring their fluctuations in the circulation as potential biomarkers inasmuch their circulating levels are increased in metabolic syndrome patients. Because of the mixed components of EVs, the content or the number of EVs derived from distinct cells of origin, the mode of cell stimulation, and the ensuing mechanisms for their production, it is difficult to attribute specific functions as drivers or biomarkers of diseases. This review reports recent data of EVs from different origins, including endothelial, smooth muscle cells, macrophages, hepatocytes, adipocytes, skeletal muscle, and finally, those from microbiota as bioeffectors of message, leading to metabolic syndrome. Depicting the complexity of the mechanisms involved in their functions reinforce the hypothesis that EVs are valid biomarkers, and they represent targets that can be harnessed for innovative therapeutic approaches. © 2017 American Heart Association, Inc.

  11. A method for analysis of lipid vesicle domain structure from confocal image data

    DEFF Research Database (Denmark)

    Husen, Peter Rasmussen; Fidorra, Matthias; Hartel, Steffen

    2012-01-01

    confocal imaging stacks. The technique involves projection of volumetric image data onto a triangulated surface mesh representation of the membrane, correction of photoselection effects and global motion of the vesicle during image acquisition and segmentation of the surface into domains using histograms....... The analysis allows for investigation of the morphology and size distribution of domains on the surface....

  12. Oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models

    NARCIS (Netherlands)

    Arntz, O.J.; Pieters, B.C.; Oliveira, M.C.; Broeren, M.G.A.; Bennink, M.B.; Vries, M. de; Lent, P.L.E.M. van; Koenders, M.I.; Berg, W.B. van den; Kraan, P.M. van der; Loo, F.A.J. van de

    2015-01-01

    SCOPE: This study shows the effect of bovine milk derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra-deficient mice and collagen-induced arthritis. METHODS AND RESULTS: BMEVs were isolated from semi-skimmed milk by ultracentrifugation and the particle size was around 100

  13. A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations

    DEFF Research Database (Denmark)

    Simonsson, Carl; Madsen, Jakob Torp; Graneli, Annette

    2011-01-01

    The growing focus on nanotechnology and the increased use of nano-sized structures, e.g. vesicles, in topical formulations has led to safety concerns. We have investigated the sensitizing capacity and penetration properties of a fluorescent model compound, rhodamine B isothiocyanate (RBITC), when...... penetration and increased formation of hapten-protein complexes in epidermis when RBITC is delivered in ethosomal formulations....

  14. Riboflavin and chlorophyll as photosensitizers in electroformed giant unilamellar vesicles as food models

    DEFF Research Database (Denmark)

    Wang, Hui Jing; Liang, Ran; du, Hui Hui

    2017-01-01

    Electroformed giant unilamellar vesicles (GUVs) were found to have optimal sizes (~10 µm average diameter) for studying effects of photosensitizers and antioxidants in lipid bilayers as food models. By using optical microscopy and digital image processing techniques, no membrane damage was found ...

  15. A two phase field model for tracking vesicle-vesicle adhesion.

    Science.gov (United States)

    Gu, Rui; Wang, Xiaoqiang; Gunzburger, Max

    2016-11-01

    A multi-phase-field model for simulating the adhesion between two vesicles is constructed. Two phase field functions are introduced to simulate each of the two vesicles. An energy model is defined which accounts for the elastic bending energy of each vesicle and the contact potential energy between the two vesicles; the vesicle volume and surface area constraints are imposed using a penalty method. Numerical results are provided to verify the efficacy of our model and to provide visual illustrations of the different types of contact. The method can be adjusted to solve endocytosis problems by modifying the bending rigidity coefficients of the two elastic bending energies. The method can also be extended to simulate multi-cell adhesions, one example of which is erythrocyte rouleaux. A comparison with laboratory observations demonstrates the effectiveness of the multi-phase field approach.

  16. Fusogenicity of Naja naja atra cardiotoxin-like basic protein on sphingomyelin vesicles containing oxidized phosphatidylcholine and cholesterol.

    Science.gov (United States)

    Kao, Pei-Hsiu; Chen, Ying-Jung; Yang, Shin-Yi; Lin, Shinne-Ren; Hu, Wan-Ping; Chang, Long-Sen

    2013-06-01

    This study investigated the effect of oxidized phosphatidylcholine (oxPC) and cholesterol (Chol) on Naja naja atra cardiotoxin-like basic protein (CLBP)-induced fusion and leakage in sphingomyelin (SM) vesicles. Compared with those on PC/SM/Chol vesicles, CLBP showed a lower activity to induce membrane permeability but a higher fusogenicity on oxPC/SM/Chol vesicles. A reduction in inner-leaflet fusion elucidated that CLBP fusogenicity was not in parallel to its membrane-leakage activity on oxPC/SM/Chol vesicles. The lipid domain formed by Chol and SM supported CLBP fusogenicity on oxPC/SM/Chol vesicles, while oxPC altered the interacted mode of CLBP with oxPC/SM/Chol vesicles as evidenced by Fourier transform infrared spectra analyses and colorimetric phospholipid/polydiacetylene membrane assay. Although CLBP showed similar binding affinity with PC/SM/Chol and oxPC/SM/Chol vesicles, the binding capability of CLBP with PC/SM/Chol and oxPC/SM/Chol vesicles was affected differently by NaCl. This emphasized that CLBP adopted different membrane interaction modes upon binding with PC/SM/Chol and oxPC/SM/Chol vesicles. CLBP induced fusion in vesicles containing oxPC bearing the aldehyde group, and aldehyde scavenger methoxyamine abrogated the CLBP ability to induce oxPC/SM/Chol fusion. Taken together, our data indicate that Chol and oxPC bearing aldehyde group alter the CLBP membrane-binding mode, leading to fusogenicity promotion while reducing the membrane-damaging activity of CLBP.

  17. Comparison of 180-degree and 90-degree needle rotation to reduce wound size in PIT-injected juvenile Chinook salmon

    Energy Technology Data Exchange (ETDEWEB)

    Bryson, Amanda J.; Woodley, Christa M.; Karls, Rhonda K.; Hall, Kathleen D.; Weiland, Mark A.; Deng, Zhiqun; Carlson, Thomas J.; Eppard, Matthew B.

    2013-04-30

    Animal telemetry, which requires the implantation of passive transponders or active transmitters, is used to monitor and assess fish stock and conservation to gain an understanding of fish movement and behavior. As new telemetry technologies become available, studies of their effects on species of interest are imperative as is development of implantation techniques. In this study, we investigated the effects of bevel rotation (0-, 90-, 180-degree axis rotation) on wound extent, tag loss, and wound healing rates in juvenile Chinook salmon injected with an 8-gauge needle, which is required for implantation of the novel injectable Juvenile Salmon Acoustic Telemetry Systems (JSATS) acoustic transmitter or large passive integrated transponder (PIT) tags. Although the injection sites were not closed after injection (e.g., with sutures or glue), there were no mortalities, dropped tags, or indications of fungus, ulceration, and/or redness around the wound. On Day 0 and post-implantation Day 7, the 90-degree bevel rotation produced smaller wound extent than the 180-degree bevel rotation. No axis rotation (0-degrees) resulted in the PIT tag frequently misleading or falling out upon injection. The results of this study indicated the 90-degree bevel rotation was the more efficient technique, produced less wound extent. Given the wound extent compared to size of fish, we recommend researchers should consider a 90-degree rotation over the 180-degree rotation in telemetry studies. Highlights •Three degrees of needle rotation were examined for effects in Chinook salmon. •Mortality, tag loss, wound extent, healing, and infection indicators were measured. •There were no mortalities, tag loss, or indications of infection. •The 90-degree needle rotation through Day 7 produced the smallest wound extent.

  18. Single-vesicle detection and analysis of peptide-induced membrane permeabilization

    DEFF Research Database (Denmark)

    Kristensen, Kasper; Ehrlich, Nicky; Henriksen, Jonas Rosager

    2015-01-01

    methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles...... of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin...

  19. Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array

    Directory of Open Access Journals (Sweden)

    Joanne Louise Welton

    2016-06-01

    Full Text Available Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen–antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios.

  20. Electroformation of Giant Vesicles on a Polymer Mesh

    Directory of Open Access Journals (Sweden)

    Yukihisa Okumura

    2011-07-01

    Full Text Available Electroformation of cell-sized lipid membrane vesicles (giant vesicles, GVs from egg yolk phosphatidylcholine under applied electric voltage was examined on a substrate of a polymer mesh placed between two planar indium tin oxide coated glass electrodes. Under appropriate conditions, GVs were formed in good yield on meshes of various polymer materials, namely, hydrophobic poly(propylene, poly(ethylene terephthalate, a carbon fiber/nylon composite, and relatively hydrophilic nylon. Arranging threads in a mesh structure with appropriate openings improved GV formation compared to simply increasing the number of threads. For optimal electroformation of GVs, the size and shape of a mesh opening were crucial. With a too large opening, GV formation deteriorated. When the sides of an opening were partially missing, GV formation did not occur efficiently. With an adequate opening, a deposited lipid solution could fill the opening, and a relatively uniform lipid deposit formed on the surface of threads after evaporation of the solvent. This could supply a sufficient amount of lipids to the opening and also prevent a lipid deposit from becoming too thick for electroformation. As a result, good GV formation was often observed in openings filled with swelled lipid.

  1. Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis.

    Science.gov (United States)

    Germain, Devon R; Li, Lei; Hildebrandt, Matthew R; Simmonds, Andrew J; Hughes, Sarah C; Godbout, Roseline

    2015-11-15

    Mammalian DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. Here, we report phenotypes associated with a null Ddx1 (Ddx1(AX)) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. We demonstrate that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. Our data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Mechanics of post-fusion exocytotic vesicle.

    Science.gov (United States)

    Stephens, Thomas; Wu, Zhanghan; Liu, Jian

    2017-05-23

    Exocytosis is an important cellular process controlled by metabolic signaling. It involves vesicle fusion to the plasma membrane, followed by the opening of a fusion pore, and the subsequent release of the vesicular lumen content into the extracellular space. While most modeling efforts focus on the events leading to membrane fusion, how the vesicular membrane remodels after fusing to plasma membrane remains unclear. This latter event dictates the nature and the efficiency of exocytotic vesicular secretions, and is thus critical for exocytotic function. We provide a generic membrane mechanical model to systematically study the fate of post-fusion vesicles. We show that while membrane stiffness favors full-collapse vesicle fusion into the plasma membrane, the intravesicular pressure swells the vesicle and causes the fusion pore to shrink. Dimensions of the vesicle and its associated fusion pore further modulate this mechanical antagonism. We systematically define the mechanical conditions that account for the full spectrum of the observed vesicular secretion modes. Our model therefore can serve as a unified theoretical framework that sheds light on the elaborate control mechanism of exocytosis.

  3. Astrocytic Vesicle Mobility in Health and Disease

    Directory of Open Access Journals (Sweden)

    Robert Zorec

    2013-05-01

    Full Text Available Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide, (ii plasma membrane exchange of transporters and receptors (EAAT2, MHC-II, and (iii the involvement of vesicle mobility carrying aquaporins (AQP4 in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  4. Mechanics of post-fusion exocytotic vesicle

    Science.gov (United States)

    Stephens, Thomas; Wu, Zhanghan; Liu, Jian

    2017-06-01

    Exocytosis is an important cellular process controlled by metabolic signaling. It involves vesicle fusion to the plasma membrane, followed by the opening of a fusion pore, and the subsequent release of the vesicular lumen content into the extracellular space. While most modeling efforts focus on the events leading to membrane fusion, how the vesicular membrane remodels after fusing to plasma membrane remains unclear. This latter event dictates the nature and the efficiency of exocytotic vesicular secretions, and is thus critical for exocytotic function. We provide a generic membrane mechanical model to systematically study the fate of post-fusion vesicles. We show that while membrane stiffness favors full-collapse vesicle fusion into the plasma membrane, the intravesicular pressure swells the vesicle and causes the fusion pore to shrink. Dimensions of the vesicle and its associated fusion pore further modulate this mechanical antagonism. We systematically define the mechanical conditions that account for the full spectrum of the observed vesicular secretion modes. Our model therefore can serve as a unified theoretical framework that sheds light on the elaborate control mechanism of exocytosis.

  5. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

    Directory of Open Access Journals (Sweden)

    A. V. Oberemko

    2014-12-01

    Full Text Available This review presents a generalized definition of vesicles as bilayer extracellular organelles of all celular forms of life: not only eu-, but also prokaryotic. The structure and composition of extracellular vesicles, history of research, nomenclature, their impact on life processes in health and disease are discussed. Moreover, vesicles may be useful as clinical instruments for biomarkers, and they are promising as biotechnological drug. However, many questions in this area are still unresolved and need to be addressed in the future. The most interesting from the point of view of practical health care represents a direction to study the effect of exosomes and microvesicles in the development and progression of a particular disease, the possibility of adjusting the pathological process by means of extracellular vesicles of a particular type, acting as an active ingredient. Relevant is the further elucidation of the role and importance of exosomes to the surrounding cells, tissues and organs at the molecular level, the prospects for the use of non-cellular vesicles as biomarkers of disease.

  6. A simple and fast method to study the hydrodynamic size difference of protein disulfide isomerase in oxidized and reduced form using gold nanoparticles and dynamic light scattering.

    Science.gov (United States)

    Zheng, Tianyu; Cherubin, Patrick; Cilenti, Lucia; Teter, Ken; Huo, Qun

    2016-02-07

    The hydrodynamic dimension of a protein is a reflection of both its molecular weight and its tertiary structures. Studying the hydrodynamic dimensions of proteins in solutions can help elucidate the structural properties of proteins. Here we report a simple and fast method to measure the hydrodyamic size of a relatively small protein, protein disulfide isomerase (PDI), using gold nanoparticle probes combined with dynamic light scattering. Proteins can readily adsorb to citrate-capped gold nanoparticles to form a protein corona. By measuring the average diameter of the gold nanoparticles before and after protein corona formation, the hydrodynamic diameter of the protein can be deduced from the net particle size increase of the assay solution. This study found that when the disulfide bonds in PDI are reduced to thiols, the reduced PDI exhibits a smaller hydrodynamic diameter than the oxided PDI. This finding is in good agreement with the X-ray diffraction analysis of PDI in single crystals. In comparison with other techniques that are used for protein hydrodynamic size analysis, the current method is easy to use, requires a trace amount of protein samples, with results obtained in minutes instead of hours.

  7. A liposome-based size calibration method for measuring microvesicles by flow cytometry

    DEFF Research Database (Denmark)

    Simonsen, Jens Bæk

    2016-01-01

    BACKGROUND: Over the last years the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized. METHODS: This work suggests to use artificial vesicles as calibrators to ascertain the size of microvesicles from the side...... to verify the correlation between the SSC from a liposome and the corresponding size. CONCLUSIONS: We show that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering...... from ~400 nm sized vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 to 200 nm dependent on the vesicle size. This article is protected by copyright. All rights reserved....

  8. Coarse-grained molecular simulations of the melting kinetics of small unilamellar vesicles.

    Science.gov (United States)

    Patel, Lara A; Kindt, James T

    2016-02-14

    Simulations of small unilamellar lipid bilayer vesicles have been performed to model their response to an instantaneous rise in temperature, starting from an initial low-temperature structure, to temperatures near or above the main chain transition temperature. The MARTINI coarse-grained force-field was used to construct slabs of gel-phase DPPC bilayers, which were assembled into truncated icosahedral structures containing 13,165 or 31,021 lipids. Equilibration at 280 K produced structures with several (5-8) domains, characterized by facets of lipids packed in the gel phase connected by disordered ridges. Instantaneous heating to final temperatures ranging from 290 K to 310 K led to partial or total melting over 500 ns trajectories, accompanied by changes in vesicle shape and the sizes and arrangements of remaining gel-phase domains. At temperatures that produced partial melting, the gel-phase lipid content of the vesicles followed an exponential decay, similar in form and timescale to the sub-microsecond phase of melting kinetics observed in recent ultrafast IR temperature-jump experiments. The changing rate of melting appears to be the outcome of a number of competing contributions, but changes in curvature stress arising from the expansion of the bilayer area upon melting are a major factor. The simulations give a more detailed picture of the changes that occur in frozen vesicles following a temperature jump, which will be of use for the interpretation of temperature-jump experiments on vesicles.

  9. Multivalent ligand-receptor-mediated interaction of small filled vesicles with a cellular membrane

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2017-07-01

    The ligand-receptor-mediated contacts of small sub-100-nm-sized lipid vesicles (or nanoparticles) with the cellular membrane are of interest in the contexts of cell-to-cell communication, endocytosis of membrane-coated virions, and drug (RNA) delivery. In all these cases, the interior of vesicles is filled by biologically relevant content. Despite the diversity of such systems, the corresponding ligand-receptor interaction possesses universal features. One of them is that the vesicle-membrane contacts can be accompanied by the redistribution of ligands and receptors between the contact and contact-free regions. In particular, the concentrations of ligands and receptors may become appreciably higher in the contact regions and their composition may there be different compared to that in the suspended state in the solution. A statistical model presented herein describes the corresponding distribution of various ligands and receptors and allows one to calculate the related change of the free energy with variation of the vesicle-engulfment extent. The results obtained are used to clarify the necessary conditions for the vesicle-assisted pathway of drug delivery.

  10. Directed vesicle transport by diffusio-osmosis

    Science.gov (United States)

    Michler, D.; Shahidzadeh, N.; Sprik, R.; Bonn, D.

    2015-04-01

    We present a study on surfactant vesicles that spontaneously move towards an oil droplet that is deposited on a glass substrate. Tracer particles in the surfactant solution show that the motion is not self-propelled: the vesicles are entrained by a macroscopic hydrodynamic flow. Measurements of the flow velocity suggest that the flow is of diffusio-osmotic nature. The surfactant is observed to move into the oil phase which creates a gradient in ion concentration in the vicinity of the droplet. As the diffusion coefficients of the surfactant's co- and counter-ions differ, a charge separation takes place and an electric field arises. This electric field then generates a hydrodynamic flow along the charged glass substrate in which the vesicles are entrained.

  11. Functionalization of Block Copolymer Vesicle Surfaces

    Directory of Open Access Journals (Sweden)

    Wolfgang Meier

    2011-01-01

    Full Text Available In dilute aqueous solutions certain amphiphilic block copolymers self-assemble into vesicles that enclose a small pool of water with a membrane. Such polymersomes have promising applications ranging from targeted drug-delivery devices, to biosensors, and nanoreactors. Interactions between block copolymer membranes and their surroundings are important factors that determine their potential biomedical applications. Such interactions are influenced predominantly by the membrane surface. We review methods to functionalize block copolymer vesicle surfaces by chemical means with ligands such as antibodies, adhesion moieties, enzymes, carbohydrates and fluorophores. Furthermore, surface-functionalization can be achieved by self-assembly of polymers that carry ligands at their chain ends or in their hydrophilic blocks. While this review focuses on the strategies to functionalize vesicle surfaces, the applications realized by, and envisioned for, such functional polymersomes are also highlighted.

  12. Vesicles formed by mixed catanionic surfactants as novel pseudostationary phase in electrokinetic chromatography.

    Science.gov (United States)

    Lu, Jie; Ni, Xinjiong; Cao, Yuhua; Ma, Xinyu; Cao, Guangqun

    2014-09-12

    In this paper, a novel pseudostationary phase (PSP), the vesicle formed from octyltriethylammonium bromide (C8NE3Br) and sodium dodecyl benzene sulfonate (SDBS), has been developed in electrokinetic chromatography (EKC). Physicochemical parameters of the mixture of catanionic surfactants such as ζ potential and size of the aggregates were characterized as the molar ratio of C8NE3Br to SDBS varied from 2:8 to 8:2 and total concentration of surfactants fixed at 20mM. At any ratio mentioned above, ζ potential of mixture of catanionic surfactants remained negative. The absolute values of ζ potential were even larger than in only SDBS system as the molar ratio of C8NE3Br to SDBS less than 4:6, and they decreased as increasing the ratio of cationic surfactants. The size of the aggregates became smaller as the ratio was close to 1. Unexpectedly, the size was smallest at ratio of 3:7 and 6:4, instead of at 5:5. Notably, coagulation did not occur in the catanionic system at any proportion of each other. TEM testified the formation of vesicles. The performance of the vesicle as PSP was evaluated by separating eight kinds of corticosteroids with EKC, these analytes were separated completely without any additives. Compared with SDS microemulsion modified with ionic liquid (IL) and polymeric micelle, the novel vesicle PSP had better separation performances. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Electrohydrodynamics of a compound vesicle under an AC electric field.

    Science.gov (United States)

    Sinha, Kumari Priti; Thaokar, Rochish M

    2017-07-12

    Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius [Formula: see text], is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

  14. Electrohydrodynamics of a compound vesicle under an AC electric field

    Science.gov (United States)

    Priti Sinha, Kumari; Thaokar, Rochish M.

    2017-07-01

    Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius λ {{R}o} , is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

  15. Isolation and characterization of platelet-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Maria T. Aatonen

    2014-08-01

    Full Text Available Background: Platelet-derived extracellular vesicles (EVs participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods: Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results: The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions: Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As

  16. CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

    Science.gov (United States)

    Kabachinski, Greg; Yamaga, Masaki; Kielar-Grevstad, D. Michelle; Bruinsma, Stephen; Martin, Thomas F. J.

    2014-01-01

    Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13. PMID:24356451

  17. Conducting EQ-5D Valuation Studies in Resource-Constrained Countries: The Potential Use of Shrinkage Estimators to Reduce Sample Size.

    Science.gov (United States)

    Chan, Kelvin K W; Xie, Feng; Willan, Andrew R; Pullenayegum, Eleanor M

    2018-01-01

    Resource-constrained countries have difficulty conducting large EQ-5D valuation studies, which limits their ability to conduct cost-utility analyses using a value set specific to their own population. When estimates of similar but related parameters are available, shrinkage estimators reduce uncertainty and yield estimators with smaller mean square error (MSE). We hypothesized that health utilities based on shrinkage estimators can reduce MSE and mean absolute error (MAE) when compared to country-specific health utilities. We conducted a simulation study (1,000 iterations) based on the observed means and standard deviations (or standard errors) of the EQ-5D-3L valuation studies from 14 counties. In each iteration, the simulated data were fitted with the model based on the country-specific functional form of the scoring algorithm to create country-specific health utilities ("naïve" estimators). Shrinkage estimators were calculated based on the empirical Bayes estimation methods. The performance of shrinkage estimators was compared with those of the naïve estimators over a range of different sample sizes based on MSE, MAE, mean bias, standard errors and the width of confidence intervals. The MSE of the shrinkage estimators was smaller than the MSE of the naïve estimators on average, as theoretically predicted. Importantly, the MAE of the shrinkage estimators was also smaller than the MAE of the naïve estimators on average. In addition, the reduction in MSE with the use of shrinkage estimators did not substantially increase bias. The degree of reduction in uncertainty by shrinkage estimators is most apparent in valuation studies with small sample size. Health utilities derived from shrinkage estimation allow valuation studies with small sample size to "borrow strength" from other valuation studies to reduce uncertainty.

  18. Hard and soft tissue changes around implants installed in regular-sized and reduced alveolar bony ridges. An experimental study in dogs.

    Science.gov (United States)

    Baffone, Gabriele; Lang, Niklaus P; Pantani, Fabio; Favero, Giovanni; Ferri, Mauro; Botticelli, Daniele

    2015-01-01

    To study bony and soft tissue changes at implants installed in alveolar bony ridges of different widths. In 6 Labrador dogs, the mandibular premolars and first molars were extracted, and a buccal defect was created in the left side at the third and fourth premolars by removing the buccal bone and the inter-radicular and interdental septa. Three months after tooth extraction, full-thickness mucoperiosteal flaps were elevated, and implants were installed, two at the reduced (test) and two at the regular-sized ridges (control). Narrow or wide abutments were affixed to the implants. After 3 months, biopsies were harvested, and ground sections prepared for histological evaluation. A higher vertical buccal bony crest resorption was found at the test (1.5 ± 0.7 mm and 1.0 ± 0.7 mm) compared to the control implants (1.0 ± 0.5 mm and 0.7 ± 0.4 mm), for both wide and narrow abutment sites. A higher horizontal alveolar resorption was identified at the control compared to the test implants. The difference was significant for narrow abutment sites. The peri-implant mucosa was more coronally positioned at the narrow abutment, in the test sites, while for the control sites, the mucosal adaptation was more coronal at the wide abutment sites. These differences, however, did not reach statistical significance. Implants installed in regular-sized alveolar ridges had a higher horizontal, but a lower vertical buccal bony crest resorption compared to implants installed in reduced alveolar ridges. Narrow abutments in reduced ridges as well as wide abutments in regular-sized ridges yielded less soft tissue recession compared to their counterparts. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Mineralization during matrix-vesicle-mediated mantle dentine formation in molars of albino rats: a microanalytical and ultrastructural study.

    Science.gov (United States)

    Stratmann, U; Schaarschmidt, K; Wiesmann, H P; Plate, U; Höhling, H J

    1996-05-01

    The purpose of this study was to elucidate the mineralization process of mantle dentine by ultrastructural and element-analytical investigation of matrix vesicles and successive stages. Upper second molars of albino rats were cryofixed and embedded in resin after freeze drying. Semithin dry sections were prepared for analyzing the calcium and phosphorus concentrations in the mineralized matrix vesicles or noduli, larger mineralized islands, and the mantle dentine. For ultrastructural studies, it was necessary to reduce section contact with hydrous fluids to a minimum in order to avoid preparation artifacts. The first mineral deposits were recognized as dot-like formations both in the interior of matrix vesicles and in association with the inner vesicle membrane. This indicated the existence of mineral nucleating sites located both at the inner membrane and at calcium-phosphate-binding macromolecules in the interior of the matrix vesicles. A significantly higher mineral content was found in mineralized matrix vesicles than in the mineralized extravesicular regions of the mineralized islands, suggesting the existence of a rapidly and densely mineralized matrix in the matrix vesicles. A significant increase in mineral content per volume proceeding from the mineralized islands to mantle dentine suggested a further increase in the density of mineral.

  20. Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer.

    Science.gov (United States)

    Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M; Carracedo, Arkaitz

    2016-08-01

    Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data integration in the field. In this review, we provide an updated view of the potential of exosomes and microvesicles as biomarkers and the available technologies for their isolation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Eviendep® reduces number and size of duodenal polyps in familial adenomatous polyposis patients with ileal pouch-anal anastomosis.

    Science.gov (United States)

    Calabrese, Carlo; Praticò, Chiara; Calafiore, Andrea; Coscia, Maurizio; Gentilini, Lorenzo; Poggioli, Gilberto; Gionchetti, Paolo; Campieri, Massimo; Rizzello, Fernando

    2013-09-14

    To evaluate if 3 mo oral supplementation with Eviendep® was able to reduce the number of duodenal polyps in familial adenomatous polyposis (FAP) patients with ileal pouch-anal anastomosis (IPAA). Eleven FAP patients with IPAA and duodenal polyps were enrolled. They underwent upper gastrointestinal (GI) endoscopy at the baseline and after 3 mo of treatment. Each patient received 5 mg Eviendep twice a day, at breakfast and dinner time, for 3 mo. Two endoscopists evaluated in a blinded manner the number and size of duodenal polyps. Upper GI endoscopies with biopsies were performed at the baseline (T0) with the assessment of the Spigelman score. Polyps > 10 mm were removed during endoscopy and at the end of the procedure a new Spigelman score was determined (T1). The procedure was repeated 3 mo after the baseline (T2). Four photograms were examined for each patient, at T1 and T2. The examined area was divided into 3 segments: duodenal bulb, second and third portion duodenum. Biopsy specimens were taken from all polyps > 10 mm and from all suspicious ones, defined by the presence of a central depression, irregular surface, or irregular vascular pattern. Histology was classified according to the updated Vienna criteria. At baseline the mean number of duodenal detected polyps was 27.7 and mean sizes were 15.8 mm; the mean Spigelman score was 7.1. After polypectomy the mean number of duodenal detected polyps was 25.7 and mean sizes were 7.6 mm; the mean Spigelman score was 6.4. After 3 mo of Eviendep bid, all patients showed a reduction of number and size of duodenal polyps. The mean number of duodenal polyps was 8 (P = 0.021) and mean size was 4.4 mm; the mean Spigelman score was 6.6. Interrater agreement was measured. Lesions > 1 cm found a very good degree of concordance (kappa 0.851) and a good concordance was as well encountered for smaller lesions (kappa 0.641). Our study demonstrated that short-term (90 d) supplementation with Eviendep® in FAP patients with IPAA

  2. Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sampathkumar Rangasamy

    2016-09-01

    Full Text Available Rett syndrome (RTT is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene, encoding methyl-CpG-binding protein 2. We have created a mouse model (Mecp2 A140V “knock-in” mutant expressing the recurrent human MECP2 A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN cultures from mutant (Mecp2A140V/y and wild type (Mecp2+/y male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele and a wild type Mecp2 gene linked to an eGFP transgene (paternal allele. In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1 treatment of neuronal cells from Mecp2 mutant mice rescued the soma size phenotype. We also found that Mecp2  mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i reduced neuronal size is an important in vitro cellular phenotype of Mecp2 mutation in mice, and ii MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, in vitro assay to screen for compounds that rescue small neuronal phenotype (“phenotypic assay”.

  3. The role of extracellular vesicles in malaria biology and pathogenesis.

    Science.gov (United States)

    Sampaio, Natalia Guimaraes; Cheng, Lesley; Eriksson, Emily M

    2017-06-09

    In the past decade, research on the functions of extracellular vesicles in malaria has expanded dramatically. Investigations into the various vesicle types, from both host and parasite origin, has revealed important roles for extracellular vesicles in disease pathogenesis and susceptibility, as well as cell-cell communication and immune responses. Here, work relating to extracellular vesicles in malaria is reviewed, and the areas that remain unknown and require further investigations are highlighted.

  4. SNAP-25 gene family members differentially support secretory vesicle fusion.

    Science.gov (United States)

    Arora, Swati; Saarloos, Ingrid; Kooistra, Robbelien; van de Bospoort, Rhea; Verhage, Matthijs; Toonen, Ruud F

    2017-06-01

    Neuronal dense-core vesicles (DCVs) transport and secrete neuropeptides necessary for development, plasticity and survival, but little is known about their fusion mechanism. We show that Snap-25 -null mutant (SNAP-25 KO) neurons, previously shown to degenerate after 4 days in vitro (DIV), contain fewer DCVs and have reduced DCV fusion probability in surviving neurons at DIV14. At DIV3, before degeneration, SNAP-25 KO neurons show normal DCV fusion, but one day later fusion is significantly reduced. To test if other SNAP homologs support DCV fusion, we expressed SNAP-23, SNAP-29 or SNAP-47 in SNAP-25 KO neurons. SNAP-23 and SNAP-29 rescued viability and supported DCV fusion in SNAP-25 KO neurons, but SNAP-23 did so more efficiently. SNAP-23 also rescued synaptic vesicle (SV) fusion while SNAP-29 did not. SNAP-47 failed to rescue viability and did not support DCV or SV fusion. These data demonstrate a developmental switch, in hippocampal neurons between DIV3 and DIV4, where DCV fusion becomes SNAP-25 dependent. Furthermore, SNAP-25 homologs support DCV and SV fusion and neuronal viability to variable extents - SNAP-23 most effectively, SNAP-29 less so and SNAP-47 ineffectively. © 2017. Published by The Company of Biologists Ltd.

  5. Lethal giant larvae 2 regulates development of the ciliated organ Kupffer's vesicle.

    Science.gov (United States)

    Tay, Hwee Goon; Schulze, Sabrina K; Compagnon, Julien; Foley, Fiona C; Heisenberg, Carl-Philipp; Yost, H Joseph; Abdelilah-Seyfried, Salim; Amack, Jeffrey D

    2013-04-01

    Motile cilia perform crucial functions during embryonic development and throughout adult life. Development of organs containing motile cilia involves regulation of cilia formation (ciliogenesis) and formation of a luminal space (lumenogenesis) in which cilia generate fluid flows. Control of ciliogenesis and lumenogenesis is not yet fully understood, and it remains unclear whether these processes are coupled. In the zebrafish embryo, lethal giant larvae 2 (lgl2) is expressed prominently in ciliated organs. Lgl proteins are involved in establishing cell polarity and have been implicated in vesicle trafficking. Here, we identified a role for Lgl2 in development of ciliated epithelia in Kupffer's vesicle, which directs left-right asymmetry of the embryo; the otic vesicles, which give rise to the inner ear; and the pronephric ducts of the kidney. Using Kupffer's vesicle as a model ciliated organ, we found that depletion of Lgl2 disrupted lumen formation and reduced cilia number and length. Immunofluorescence and time-lapse imaging of Kupffer's vesicle morphogenesis in Lgl2-deficient embryos suggested cell adhesion defects and revealed loss of the adherens junction component E-cadherin at lateral membranes. Genetic interaction experiments indicate that Lgl2 interacts with Rab11a to regulate E-cadherin and mediate lumen formation that is uncoupled from cilia formation. These results uncover new roles and interactions for Lgl2 that are crucial for both lumenogenesis and ciliogenesis and indicate that these processes are genetically separable in zebrafish.

  6. Lethal giant larvae 2 regulates development of the ciliated organ Kupffer’s vesicle

    Science.gov (United States)

    Tay, Hwee Goon; Schulze, Sabrina K.; Compagnon, Julien; Foley, Fiona C.; Heisenberg, Carl-Philipp; Yost, H. Joseph; Abdelilah-Seyfried, Salim; Amack, Jeffrey D.

    2013-01-01

    Motile cilia perform crucial functions during embryonic development and throughout adult life. Development of organs containing motile cilia involves regulation of cilia formation (ciliogenesis) and formation of a luminal space (lumenogenesis) in which cilia generate fluid flows. Control of ciliogenesis and lumenogenesis is not yet fully understood, and it remains unclear whether these processes are coupled. In the zebrafish embryo, lethal giant larvae 2 (lgl2) is expressed prominently in ciliated organs. Lgl proteins are involved in establishing cell polarity and have been implicated in vesicle trafficking. Here, we identified a role for Lgl2 in development of ciliated epithelia in Kupffer’s vesicle, which directs left-right asymmetry of the embryo; the otic vesicles, which give rise to the inner ear; and the pronephric ducts of the kidney. Using Kupffer’s vesicle as a model ciliated organ, we found that depletion of Lgl2 disrupted lumen formation and reduced cilia number and length. Immunofluorescence and time-lapse imaging of Kupffer’s vesicle morphogenesis in Lgl2-deficient embryos suggested cell adhesion defects and revealed loss of the adherens junction component E-cadherin at lateral membranes. Genetic interaction experiments indicate that Lgl2 interacts with Rab11a to regulate E-cadherin and mediate lumen formation that is uncoupled from cilia formation. These results uncover new roles and interactions for Lgl2 that are crucial for both lumenogenesis and ciliogenesis and indicate that these processes are genetically separable in zebrafish. PMID:23482490

  7. Adsorption of DOPC vesicles on hydrophobic substrates in the ...

    Indian Academy of Sciences (India)

    Administrator

    In the present study, the interaction between an intact DOPC vesicle and the hydropho- bic surface is mainly through van der Waals interac- tion. In presence of increasing concentrations of electrolytes, counter ions are present in the vicinity of the DOPC vesicle. As the vesicle approaches the solid substrate, the counter ions ...

  8. Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

    Science.gov (United States)

    Farsi, Zohreh; Preobraschenski, Julia; van den Bogaart, Geert; Riedel, Dietmar; Jahn, Reinhard; Woehler, Andrew

    2016-02-26

    Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. Copyright © 2016, American Association for the Advancement of Science.

  9. Role of Outer Membrane Vesicles of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 8. Role of Outer Membrance Vesicles of Bacteria. M V Jagannadham M K Chattopadhyay. General Article Volume 20 Issue 8 ... Keywords. Outer membrane ves ic les (OMVs); secretion; communication; virulence; antibiotic resistance; vaccines.

  10. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    Directory of Open Access Journals (Sweden)

    David R Stevens

    2011-02-01

    Full Text Available The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP and a slowly releasable (SRP pool are followed by sustained release, due to maturation and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles.

  11. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  12. Compartmentalization and Transport in Synthetic Vesicles

    Directory of Open Access Journals (Sweden)

    Christine eSchmitt

    2016-02-01

    Full Text Available Nano-scale vesicles have become a popular tool in life sciences. Besides liposomes that are generated from phospholipids of natural origin, polymersomes fabricated of synthetic block copolymers enjoy increasing popularity, as they represent more versatile membrane building blocks that can be selected based on their specific physicochemical properties, like permeability, stability or chemical reactivity.In this review, we focus on the application of simple and nested artificial vesicles in synthetic biology. First, we provide an introduction into the utilization of multi-compartmented vesosomes as compartmentalized nano-scale bioreactors. In the bottom-up development of protocells from vesicular nano-reactors, the specific exchange of pathway intermediates across compartment boundaries represents a bottleneck for future studies. To date, most compartmented bioreactors rely on unspecific exchange of substrates and products. This is either based on changes in permeability of the coblock polymer shell by physicochemical triggers or by the incorporation of unspecific porin proteins into the vesicle membrane. Since the incorporation of membrane transport proteins into simple and nested artificial vesicles offers the potential for specific exchange of substances between subcompartments, it opens new vistas in the design of protocells. Therefore we devote the main part of the review to summarize the technical advances in the use of phospholipids and block copolymers for the reconstitution of membrane proteins.

  13. Functional transferred DNA within extracellular vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Jin [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Jiangsu Province (China); Wu, Gengze [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Jose, Pedro A. [Division of Nephrology, Department of Medicine and Physiology, University of Maryland, School of Medicine, Baltimore, MD 21201 (United States); Zeng, Chunyu, E-mail: Chunyuzeng01@163.com [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China)

    2016-11-15

    Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

  14. Theory of Disk-to-Vesicle Transformation

    Science.gov (United States)

    Li, Jianfeng; Shi, An-Chang

    2009-03-01

    Self-assembled membranes from amphiphilic molecules, such as lipids and block copolymers, can assume a variety of morphologies dictated by energy minimization of system. The membrane energy is characterized by a bending modulus (κ), a Gaussian modulus (κG), and the line tension (γ) of the edge. Two basic morphologies of membranes are flat disks that minimize the bending energy at the cost of the edge energy, and enclosed vesicles that minimize the edge energy at the cost of bending energy. In our work, the transition from disk to vesicle is studied theoretically using the string method, which is designed to find the minimum energy path (MEP) or the most probable transition path between two local minima of an energy landscape. Previous studies of disk-to-vesicle transition usually approximate the transitional states by a series of spherical cups, and found that the spherical cups do not correspond to stable or meta-stable states of the system. Our calculation demonstrates that the intermediate shapes along the MEP are very different from spherical cups. Furthermore, some of these transitional states can be meta-stable. The disk-to-vesicle transition pathways are governed by two scaled parameters, κG/κ and γR0/4κ, where R0 is the radius of the disk. In particular, a meta-stable intermediate state is predicted, which may correspond to the open morphologies observed in experiments and simulations.

  15. Characterization of Extracellular Vesicles using Raman Spectroscopy

    NARCIS (Netherlands)

    Lee, Wooje; Nanou, Afroditi; Terstappen, Leonardus Wendelinus Mathias Marie; Rho, Hoon Suk; le Gac, Severine; Offerhaus, Herman L.

    2017-01-01

    In this research, we aim to characterize extracellular vesicles(EVs) with Confocal Raman spectroscopy to reveal relevant spectral lines that signify differences between EVs derived from different cell lines. In the first stage we performed confocal Raman measurements on various EV samples. For these

  16. A novel reduction approach to fabricate quantum-sized SnO₂-conjugated reduced graphene oxide nanocomposites as non-enzymatic glucose sensors.

    Science.gov (United States)

    Ye, Yixing; Wang, Panpan; Dai, Enmei; Liu, Jun; Tian, Zhenfei; Liang, Changhao; Shao, Guosheng

    2014-05-21

    Quantum-sized SnO2 nanocrystals can be well dispersed on reduced graphene oxide (rGO) nanosheets through a convenient one-pot in situ reduction route without using any other chemical reagent or source. Highly reactive metastable tin oxide (SnO(x)) nanoparticles (NPs) were used as reducing agents and composite precursors derived by the laser ablation in liquid (LAL) technique. Moreover, the growth and phase transition of LAL-induced SnO(x) NPs and graphene oxide (GO) were examined by optical absorption, X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectroscopy and high-resolution transmission electron microscopy. Highly dispersed SnO(x) NPs can also prevent rGO from being restacked into a multilayer structure during GO reduction. Given the good electron transfer ability and unsaturated dangling bonds of rGO, as well as the ample electrocatalytic active sites of quantum-sized SnO2 NPs on unfolded rGO sheets, the fabricated SnO2-rGO nanocomposite exhibited excellent performance in the non-enzymatic electrochemical detection of glucose molecules. The use of LAL-induced reactive NPs for in situ GO reduction is also expected to be a universal and environmentally friendly approach for the formation of various rGO-based nanocomposites.

  17. Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

    NARCIS (Netherlands)

    Kok, JW; Babia, T; Klappe, K; Egea, G; Hoekstra, D

    1998-01-01

    Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degrees C, or in streptolysin O-permeabilized cells by

  18. Carbohydrate-directed synthesis of silver and gold nanoparticles: effect of the structure of carbohydrates and reducing agents on the size and morphology of the composites.

    Science.gov (United States)

    Shervani, Zameer; Yamamoto, Yasushi

    2011-04-01

    A monosaccharide (β-D-glucose) and polysaccharide (soluble starch) were used as structure directing and subsequently stabilizing agents for the synthesis of spherical nanoparticles (NPs) and nanowires of silver and gold. Homogeneous monodispersed Ag(0) nanoparticles (Ag NPs) of 15 nm diameter were obtained when 10(-4)M AgNO(3) precursor salt was reduced in starch (1 wt%)-water gel by 1 wt% β-D-glucose. For a second preparation the effect of reducing agents on the synthesis of Au(0) metallic nanoparticles (Au NPs) of 2 × 10(-4)M concentration prepared in a β-D-glucose (0.03 M)-water dispersion was studied first in detail. Different equivalent amounts of NaBH(4) and a number of pH values were evaluated for the reduction of the Au salt HAuCl(4)·3H(2)O to obtain Au NPs. The type and the amount of reducing agent, as well as the pH of the solution was shown to affect the size and morphology of the NPs. NaBH(4) (4 equiv) produced the smallest (5.3 nm (σ 0.7)) metallic particles compared to larger particles (10.0 nm (σ 1.4)) when the salt was reduced by 1 equiv of NaBH(4). Addition of excess NaBH(4) caused the NPs to settle out as a precipitate forming a mesh or wire structure rather than monodispersed particles. Low pH (pH 6) resulted in incomplete reduction, while at pH 8 the salt was completely reduced. When the salt was reduced by NaOH at pH 8, the particles were larger (14.2 nm) and less homogeneous (σ 2.8) compared to those from NaBH(4) reduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Buoyancy Limitation of Filamentous Cyanobacteria under Prolonged Pressure due to the Gas Vesicles Collapse

    Science.gov (United States)

    Abeynayaka, Helayaye Damitha Lakmali; Asaeda, Takashi; Kaneko, Yasuko

    2017-08-01

    Freshwater cyanobacterium Pseudanabaena galeata were cultured in chambers under artificially generated pressures, which correspond to the hydrostatic pressures at deep water. Variations occurred in gas vesicles volume, and buoyancy state of cells under those conditions were analyzed at different time intervals (5 min, 1 day, and 5 days). Variations in gas vesicles morphology of cells were observed by transmission electron microscopy images. Settling velocity ( Vs) of cells which governs the buoyancy was observed with the aid of a modified optical microscope. Moreover, effects of the prolonged pressure on cell ballast composition (protein and polysaccharides) were examined. Elevated pressure conditions reduced the cell ballast and caused a complete disappearance of gas vesicles in Pseudanabaena galeata cells. Hence cyanobacteria cells were not able to float within the study period. Observations and findings of the study indicate the potential application of hydrostatic pressure, which naturally occurred in hypolimnion of lakes, to inhibit the re-suspension of cyanobacteria cells.

  20. Rab3 proteins involved in vesicle biogenesis and priming in embryonic mouse chromaffin cells

    DEFF Research Database (Denmark)

    Schonn, Jean-Sébastien; van Weering, Jan R T; Mohrmann, Ralf

    2010-01-01

    The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here we used a quadruple Rab3 knock-out (rab3a, rab3b, rab3c, rab3d null, here denoted ABCD(-/-)) mouse line to investigate Rab3 function in embryonic mouse adrenal...... chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense core vesicles (LDCVs) are less abundant while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces...... rate after an initial delay. Rescue experiments showed that short-term (4-6 hours) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV...

  1. Synaptic vesicle dynamic changes in a model of fragile X.

    Science.gov (United States)

    Broek, Jantine A C; Lin, Zhanmin; de Gruiter, H Martijn; van 't Spijker, Heleen; Haasdijk, Elize D; Cox, David; Ozcan, Sureyya; van Cappellen, Gert W A; Houtsmuller, Adriaan B; Willemsen, Rob; de Zeeuw, Chris I; Bahn, Sabine

    2016-01-01

    Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.

  2. The Origin of Nanoscopic Grooving on Vesicle Walls in Submarine Basaltic Glass: Implications for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Jason E. French

    2009-01-01

    Full Text Available Dendritic networks of nanoscopic grooves measuring 50–75 nm wide by <50 nm deep occur on the walls of vesicles in the glassy margins of mid-ocean ridge pillow basalts worldwide. Until now, their exact origin and significance have remained unclear. Here we document examples of such grooved patterns on vesicle walls in rocks from beneath the North Atlantic Ocean, and give a fluid mechanical explanation for how they formed. According to this model, individual nanogrooves represent frozen viscous fingers of magmatic fluid that were injected into a thin spheroidal shell of hot glass surrounding each vesicle. The driving mechanism for this process is provided by previous numerical predictions of tangential tensile stress around some vesicles in glassy rocks upon cooling through the glass transition. The self-assembling nature of the dendritic nanogrooves, their small size, and overall complexity in form, are interesting from the standpoint of exploring new applications in the field of nanotechnology. Replicating such structures in the laboratory would compete with state-of-the-art nanolithography techniques, both in terms of pattern complexity and size, which would be useful in the fabrication of a variety of grooved nanodevices. Dendritic nanogrooving in SiO2 glass might be employed in the manufacturing of integrated circuits.

  3. Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

    Directory of Open Access Journals (Sweden)

    Bartosz Różycki

    Full Text Available The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

  4. Bile Salt Micelles and Phospholipid Vesicles Present in Simulated and Human Intestinal Fluids

    DEFF Research Database (Denmark)

    Elvang, Philipp A; Hinna, Askell H; Brouwers, Joachim

    2016-01-01

    was to apply asymmetrical flow field-flow fractionation (AF4) in combination with multiangle laser light scattering in an attempt to reveal coexistence of colloidal particles in both artificial and aspirated HIFs and to determine their sizes. Asymmetrical flow field-flow fractionation/multiangle laser light...... sizes of approximately 50 and 200 nm, respectively (intensity-weighted mean diameter, Dz), likely representing mixed cholate/phospholipid micelles and phospholipid vesicles, respectively. The sizes of the smaller 2 fractions being below the size range of multiangle laser light scattering analysis (

  5. CD18-mediated adhesion is required for the induction of a proinflammatory phenotype in lung epithelial cells by mononuclear cell-derived extracellular vesicles.

    Science.gov (United States)

    Neri, Tommaso; Scalise, Valentina; Passalacqua, Ilaria; Giusti, Ilaria; Lombardi, Stefania; Balia, Cristina; D'Alessandro, Delfo; Berrettini, Stefano; Pedrinelli, Roberto; Paggiaro, Pierluigi; Dolo, Vincenza; Celi, Alessandro

    2018-02-21

    Extracellular vesicles are submicron vesicles that upregulate the synthesis of proinflammatory mediators by lung epithelial cells. We investigated whether these structures adhere to lung epithelial cells, and whether adhesion is a prerequisite for their proinflammatory activity. Extracellular vesicles were generated by stimulation of normal human mononuclear cells with the calcium ionophore A23187, and labelled with carboxyfluorescein diacetate succinimidyl ester. Adhesion of vesicles to monolayers of immortalized bronchial epithelial (16HBE) and alveolar (A549) cells was analyzed by fluorescence microscopy. The role of candidate adhesion receptors was evaluated with inhibitory monoclonal antibodies and soluble peptides. The synthesis of proinflammatory mediators was assessed by ELISA. Transmission electron microscopy confirmed the generation of closed vesicles with an approximate size range between 50 and 600 nm. Adhesion of extracellular vesicles to epithelial cells was upregulated upon stimulation of the latter with tumor necrosis factor-α. Adhesion was blocked by an anti-CD18 antibody, by peptides containing the sequence RGD and, to a lesser extent, by an antibody to ICAM-1. The same molecules also blocked the upregulation of the synthesis of interleukin-8 and monocyte chemotactic protein-1 induced by extracellular vesicles. CD18-mediated adhesion of extracellular vesicles is a prerequisite for their proinflammatory activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

    Science.gov (United States)

    Chiang, Hsueh-Cheng; Shin, Wonchul; Zhao, Wei-Dong; Hamid, Edaeni; Sheng, Jiansong; Baydyuk, Maryna; Wen, Peter J.; Jin, Albert; Momboisse, Fanny; Wu, Ling-Gang

    2014-02-01

    Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

  7. Genetically Controlled Fusion, Exocytosis and Fission of Artificial Vesicles

    DEFF Research Database (Denmark)

    Bönzli, Eva; Hadorn, Maik; De Lucrezia, Davide

    if a special class of viral proteins, termed fusogenic peptides, were added to the external medium. In the present work, we intend to develop genetically controlled fusion, fission and exocytosis of vesicles by the synthesis of peptides within vesicles. First, we enclosed synthesized peptides in vesicles...... to induce in a next step fusion of adjacent vesicles, fission and exocytosis of nested vesicles. Second, we will replace the peptides by an enclosed cell-free expression system to internally synthesize fusion peptides. To control the gene expression, different mechanisms are available, e.g. addition...... fusion, fission and exocytosis....

  8. Loading of Vesicles into Soft Amphiphilic Nanotubes using Osmosis.

    Science.gov (United States)

    Erne, Petra M; van Bezouwen, Laura S; Štacko, Peter; van Dijken, Derk Jan; Chen, Jiawen; Stuart, Marc C A; Boekema, Egbert J; Feringa, Ben L

    2015-12-07

    The facile assembly of higher-order nanoarchitectures from simple building blocks is demonstrated by the loading of vesicles into soft amphiphilic nanotubes using osmosis. The nanotubes are constructed from rigid interdigitated bilayers which are capped with vesicles comprising phospholipid-based flexible bilayers. When a hyperosmotic gradient is applied to these vesicle-capped nanotubes, the closed system loses water and the more flexible vesicle bilayer is pulled inwards. This leads to inclusion of vesicles inside the nanotubes without affecting the tube structure, showing controlled reorganization of the self-assembled multicomponent system upon a simple osmotic stimulus. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Coated vesicles as protein release mechanism in myeloma cells.

    Science.gov (United States)

    Trombetta, L D; Lazarus, S S

    An electron microscopic study was undertaken of the protein release mechanism within myeloma cells showing a very high degree of protein production. Smooth surfaced vesicles (50 millimicrons) were seen to originate from the outer margin of the perinuclear cistern. Similar vesicles were also associated with distended Golgi sacs. Possible function of these vesicles could not be determined. Coated vesicles (60 millimicrons) originated as evaginations from endoplasmic reticulum in the transitional region. They were present throughout the cytoplasm and were seen to fuse with the cell membrane discharging an electron dense material. These vesicles are, therefore, thought to transport protein from the rough endoplasmic reticulum and discharge it at the cell surface.

  10. Sodium dodecyl sulfate/β-cyclodextrin vesicles embedded in chitosan gel for insulin delivery with pH-selective release

    Directory of Open Access Journals (Sweden)

    Zhuo Li

    2016-07-01

    Full Text Available In an answer to the challenge of enzymatic instability and low oral bioavailability of proteins/peptides, a new type of drug-delivery vesicle has been developed. The preparation, based on sodium dodecyl sulfate (SDS and β-cyclodextrin (β-CD embedded in chitosan gel, was used to successfully deliver the model drug-insulin. The self-assembled SDS/β-CD vesicles were prepared and characterized by particle size, zeta potential, appearance, microscopic morphology and entrapment efficiency. In addition, both the interaction of insulin with vesicles and the stability of insulin loaded in vesicles in the presence of pepsin were investigated. The vesicles were crosslinked into thermo-sensitive chitosan/β-glycerol phosphate solution for an in-situ gel to enhance the dilution stability. The in vitro release characteristics of insulin from gels in media at different pH values were investigated. The insulin loaded vesicles–chitosan hydrogel (IVG improved the dilution stability of the vesicles and provided pH-selective sustained release compared with insulin solution–chitosan hydrogel (ISG. In vitro, IVG exhibited slow release in acidic solution and relatively quick release in neutral solutions to provide drug efficacy. In simulated digestive fluid, IVG showed better sustained release and insulin protection properties compared with ISG. Thus IVG might improve the stability of insulin during its transport in vivo and contribute to the bioavailability and therapeutic effect of insulin.

  11. The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin.

    Science.gov (United States)

    Kristanc, Luka; Božič, Bojan; Gomišček, Gregor

    2014-10-01

    The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membrane's molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membrane's expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Demonstration of two distributions of vesicle radius in the dopamine neuron of Planorbis corneus from electrochemical data.

    Science.gov (United States)

    Anderson, B B; Chen, G; Gutman, D A; Ewing, A G

    1999-05-01

    An electrochemical model to calculate the relative size and neurotransmitter concentration of individual nerve cell vesicles is presented to examine potentially different types of vesicles in Planorbis corneus. Amperometric current transients resulting from individual exocytosis events detected from single cells contain the information necessary to quantify vesicular neurotransmitter amount and to estimate other important cellular properties such as vesicular neurotransmitter concentration and vesicle radius. Use of a simplifying assumption that the cross-sectional area of the contents of each release event is the apparent electroactive area of the electrode and that the shape of the decreasing phase of each current transient follows Cottrell-like behavior, the Cottrell equation and Faraday's law can be combined to yield expressions for relative vesicle radius and neurotransmitter concentration. This analysis has been applied to data obtained from the cell body of the giant dopamine neuron of the pond snail P. corneus. The histogram of vesicular dopamine concentration reveals a single wide distribution and the histogram of vesicle radius reveals a bimodal radius distribution. These data strongly suggest two distinct classes of vesicle radius in the P. corneus neuron lead to the bimodal distribution of amount released reported earlier.

  13. Extracellular vesicle-mediated transfer of long non-coding RNA ROR modulates chemosensitivity in human hepatocellular cancer.

    Science.gov (United States)

    Takahashi, Kenji; Yan, Irene K; Kogure, Takayuki; Haga, Hiroaki; Patel, Tushar

    2014-01-01

    Hepatocellular cancers (HCC) are highly resistant to chemotherapy. TGFβ has been associated with chemoresistance in some human cancers but the mechanisms involved are unknown. We explored how TGFβ might contribute to altered responses to therapy by assessing the involvement and mechanistic contribution of extracellular vesicle long non-coding RNA (lncRNA) in mediating TGFβ-dependent chemoresistance. TGFβ reduced the sensitivity of HCC cells to sorafenib or doxorubicin and altered the release of both extracellular vesicles and of selected lncRNA within these vesicles. Amongst these, lincRNA-ROR (linc-ROR), a stress-responsive lncRNA was highly expressed in HCC cells and enriched within extracellular vesicles derived from tumor cells. Incubation with HCC-derived extracellular vesicles increased linc-ROR expression and reduced chemotherapy-induced cell death in recipient cells. Sorafenib increased linc-ROR expression in both tumor cells and extracellular vesicles, whereas siRNA to linc-ROR increased chemotherapy-induced apoptosis and cytotoxicity. Tumor-initiating cells that express CD133 have an increased resistance to therapy. TGFβ increased expression of CD133+ cells and colony growth in limiting dilution assays, both of which were attenuated by linc-ROR knockdown. These data provide mechanistic insights into primary chemoresistance in HCC by showing that: (a) TGFβ selectively enriches linc-RoR within extracellular vesicles, which has a potential role in intercellular signaling in response to TGFβ; (b) expression and enrichment of linc-ROR during chemotherapeutic stress plays a functional role in chemoresistance; and (c) the effects of TGFβ on chemoresistance in HCC may involve linc-RoR-dependent effects on tumor-initiating cells. These findings implicate extracellular vesicle lncRNA as mediators of the chemotherapeutic response, and support targeting linc-ROR to enhance chemosensitivity in HCC.

  14. Interaction of insulin with SDS/CTAB catanionic Vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Tah, Bidisha; Pal, Prabir; Talapatra, G.B., E-mail: spgbt@iacs.res.in

    2014-01-15

    In the present study, a novel method was used for entrapping the protein, insulin into the catanionic SDS/CTAB vesicle membrane. The anionic SDS and cationic CTAB formed catanionic vesicles at particular concentration (35:65 by volume). In this study, vesicle membrane can be considered as model membrane. The vesicle formation and entrapment efficiency depend on the pH of the aqueous solution. The insulin molecules have attached with the vesicular membrane at pH 7.0. However, at acidic pH, the vesicles were ruptured and the insulin did not entrap into the vesicle membrane, whereas at alkaline pH insulin became fibriller. The scanning electron microscope (SEM), Dynamic light scattering (DLS), and Zeta potential studies established the self-assembled structure formation of insulin and catanionic vesicles. To know the protein confirmations, Circular dichroism (CD) was also employed. The temperature dependent steady state and time resolved emission spectroscopy show that at room temperature (25 °C), apart from the 305 nm tyrosine fluorescence, a new emission peak at 450 nm was observed only in case of insulin-vesicle system, and was assigned as the tyrosine phosphorescence. This phosphorescence peak is the signature of the entrapment of insulin into the vesicle membrane. Highlights: • SDS-CTAB based catanionic vesicle has been fabricated. • Insulin has been successfully immobilized on these vesicles. • Immobilized insulin shows room temperature phosphorescence.

  15. AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis.

    Science.gov (United States)

    Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

    2013-03-05

    The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI:http://dx.doi.org/10.7554/eLife.00190.001.

  16. Bicarbonate sulfate exchange in canalicular rat liver plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I. (University Hospital, Zurich (Switzerland))

    1987-10-01

    The mechanism(s) and driving forces for biliary excretion of sulfate were investigated in canalicular rat liver plasma membrane vesicles (cLPM). Incubation of cLPM vesicles in the presence of an inside-to-outside (in, out) bicarbonate gradient but not pH or out-to-in sodium gradients, stimulated sulfate uptake 10-fold compared with the absence of bicarbonate and approximately 2-fold above sulfate equilibrium (overshoot). Initial rates of this bicarbonate gradient-driven ({sup 35}S)-sulfate uptake were saturable with increasing concentrations of sulfate and could be inhibited by probenecid, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate, acetazolamide, furosemide, 4-acetamideo-4{prime}-isothiocyanostilbene-2,2{prime}-disulfonic acid, and 4,4{prime}-diisothiocyanostilbene-2,2{prime}-disulfonic acid (IC{sub 50}, {approximately}40 {mu}M). Cisinhibition of initial bicarbonate gradient-stimulated sulfate uptake and transstimulation of sulfate uptake in the absence of bicarbonate were observed with sulfate, thiosulfate, and oxalate but not with chloride, nitrate, phosphate, acetate, lactate, glutamate, aspartate, cholate, taurocholate, dehydrocholate, taurodehydrocholate, and reduced or oxidized glutathione. These findings indicate the presence of a sulfate (oxalate)-bicarbonate anion exchange system in canalicular rat liver plasma membranes. These findings support the concept that bicarbonate-sensitive transport system might play an important role in bile acid-independent canalicular bile formation.

  17. Extracellular vesicles in multiple sclerosis: what are they telling us?

    Directory of Open Access Journals (Sweden)

    Matías eSáenz-Cuesta

    2014-03-01

    Full Text Available Extracellular vesicles (EVs are membrane-bound particles secreted by almost all cell types. They are classified depending on their biogenesis and size into exosomes and microvesicles or according to their cell origin. EVs play a role in cell-to-cell communication, including contact-free cell synapsis, carrying active membrane proteins, lipids, and genetic material both inside the particle and on their surface. They have been related to several physiological and pathological conditions. In particular, increasing concentrations of EVs have been found in many autoimmune diseases including multiple sclerosis (MS. MS is a central nervous system demyelinating disease characterized by relapsing of symptoms followed by periods of remission. Close interaction between endothelial cells, leukocytes, monocytes and cells from central nervous system is crucial for the development of MS. This review summarizes the pathological role of EVs in MS and the relationship of EVs with clinical characteristics, therapy and biomarkers of the disease.

  18. Machine Learning classification of MRI features of Alzheimer's disease and mild cognitive impairment subjects to reduce the sample size in clinical trials.

    Science.gov (United States)

    Escudero, Javier; Zajicek, John P; Ifeachor, Emmanuel

    2011-01-01

    There is a need for objective tools to help clinicians to diagnose Alzheimer's Disease (AD) early and accurately and to conduct Clinical Trials (CTs) with fewer patients. Magnetic Resonance Imaging (MRI) is a promising AD biomarker but no single MRI feature is optimal for all disease stages. Machine Learning classification can address these challenges. In this study, we have investigated the classification of MRI features from AD, Mild Cognitive Impairment (MCI), and control subjects from ADNI with four techniques. The highest accuracy rates for the classification of controls against ADs and MCIs were 89.2% and 72.7%, respectively. Moreover, we used the classifiers to select AD and MCI subjects who are most likely to decline for inclusion in hypothetical CTs. Using the hippocampal volume as an outcome measure, we found that the required group sizes for the CTs were reduced from 197 to 117 AD patients and from 366 to 215 MCI subjects.

  19. "Pistacia lentiscus L." reduces the infarct size in normal fed anesthetized rabbits and possess antiatheromatic and hypolipidemic activity in cholesterol fed rabbits.

    Science.gov (United States)

    Andreadou, Ioanna; Mitakou, Sofia; Paraschos, Sotirios; Efentakis, Panagiotis; Magiatis, Prokopios; Kaklamanis, Loukas; Halabalaki, Maria; Skaltsounis, Leandros; Iliodromitis, Efstathios K

    2016-10-15

    The aim of the present study was to evaluate in vivo the potential anti-ischemic and antiatheromatic activity of Chios Mastic gum, the resin of the trunk and branches of "Pistacia lentiscus var. chia", used since antiquity in traditional Greek medicine. The main compounds of mastic are triterpenes, possessing phytosterol-like structures. This led to the hypothesis that mastic and particularly its neutral fraction, enriched in phytosterol-like compounds, possess antiatheromatic activities. Total Mastic Extract without Polymer (TMEWP) and the neutral mastic fraction (NMF) were administered orally for 6 weeks to normal fed and to cholesterol fed rabbits in the form of sunflower oil solution. All the animals were randomly divided into 6 groups, anesthetized and subjected to 30min ischemia of the heart, followed by 3h reperfusion: At the end of the experiment the area at risk and the infarct zone were determined with the aid of fluorescent particles and triphenyl tetrazolium chloride staining, and small segments of the ascending and descending aorta and the heart were taken for histologic examination. Blood samples were collected at different time points of ischemia and reperfusion, for malondialdehyde (MDA) evaluation as an index of lipid peroxidation, for total and LDL cholesterol determination and for evaluation of oxidized LDL. In the normal fed animals the NMF and the TMEWP reduced significantly the infarct size, while in the hypercholesterolemic rabbits both treatments were ineffective. Atherosclerosis was detected in all the animals fed cholesterol enriched diet in the form of subintimal accumulation of lipids and foamy macrophages. There was no detection of atherosclerosis in Groups treated with TMEWP and NMF, which both reduced the total cholesterol levels by 47 and 88% respectively, whilst had not effect on LDL oxidation. TMEWP and NMF reduced the MDA concentration in normal fed rabbits, but had no effect on MDA levels in cholesterol fed animals. TMEWP and

  20. Texture-Based Differences in Eating Rate Reduce the Impact of Increased Energy Density and Large Portions on Meal Size in Adults.

    Science.gov (United States)

    McCrickerd, Keri; Lim, Charlotte Mh; Leong, Claudia; Chia, Edwin M; Forde, Ciaran G

    2017-06-01

    Background: Large portions and high dietary energy density promote overconsumption at meal times. This could be reduced by eating slowly.Objective: Two studies investigated whether texture-based reductions in eating rate and oral processing moderate consumption at breakfast in combination with variations in energy density and portion size.Methods: Adults attended 4 breakfast sessions (2 × 2 repeated-measures design) to consume rice porridge, combining a 45% reduction in eating rate [thin porridge (140 g/min) compared with thick porridge (77 g/min)] with a 77% increase in energy density (0.57 compared with 1.01 kcal/g) in study 1 [n = 61; aged 21-48 y; body mass index (BMI; in kg/m(2)): 16-29] and a 50% increase in portion size (100% compared with 150%) in study 2 (n = 53; aged 21-42 y; BMI: 16-29). Oral processing behaviors were coded by using webcams. Porridge intake was measured alongside changes in rated appetite.Results: Increases in energy density and portion size led to increases of 80% and 13% in energy intake at breakfast, respectively (P energy intake compared with the thin versions (P energy was consumed when the thick "slow" porridge was served with a lower energy density or smaller portion (P eat in response to external features of the food environment. An opportunity exists to use a combination of energy-density dilution, smaller portions, and natural variations in food texture to design meals that promote reductions in energy intake while maintaining satiety. © 2017 American Society for Nutrition.

  1. Formulation by design based risperidone nano soft lipid vesicle as a new strategy for enhanced transdermal drug delivery: In-vitro characterization, and in-vivo appraisal.

    Science.gov (United States)

    Imam, Syed Sarim; Ahad, Abdul; Aqil, Mohammed; Akhtar, Mohd; Sultana, Yasmin; Ali, Asgar

    2017-06-01

    The present study was designed to formulate and optimize transdermal risperidone soft lipid vesicles. The formulation optimized with phospholipid, safranal and ethanol were incorporated as permeation and absorption enhancers. The optimized risperidone soft lipid vesicle was further evaluated for skin irritation study, in-vivo pharmacokinetic study and locomotor activity. Three factor three level Box-Behnken design (BBD) was used to statistically optimize soft lipid vesicle using safranal (A), ethanol (B)and phospholipid (C) as independent variable, while their effect was observed for vesicle size (Y 1 ), entrapment efficiency (Y 2 ) and flux (Y 3 ). The optimized risperidone soft lipid vesicle (Ris-opt) showed nanometric vesicle size, high entrapment efficiency and marked enhancement in transdermal flux. The extent of absorption from Ris-opt was greater when compared to oral suspension with relative bioavailability of 177%. The histopathological evaluation revealed developed formulation did not showed skin irritation compared to standard irritant. The significant findings presented here encourage further studies with risperidone soft lipid vesicles for treatment of schizophrenia. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Ternary phase behaviour and vesicle formation of a sodium N-lauroylsarcosinate hydrate/1-decanol/water system

    Science.gov (United States)

    Akter, Nasima; Radiman, Shahidan; Mohamed, Faizal; Rahman, Irman Abdul; Reza, Mohammad Imam Hasan

    2011-08-01

    The phase behaviour of a system composed of amino acid-based surfactant (sodium N-lauroylsarcosinate hydrate), 1-decanol and deionised water was investigated for vesicle formation. Changing the molar ratio of the amphiphiles, two important aggregate structures were observed in the aqueous corner of the phase diagram. Two different sizes of microemulsions were found at two amphiphile-water boundaries. A stable single vesicle lobe was found for 1∶2 molar ratios in 92 wt% water with vesicles approximately 100 nm in size and with high zeta potential value. Structural variation arises due to the reduction of electrostatic repulsions among the ionic headgroups of the surfactants and the hydration forces due to adsorbed water onto monolayer's. The balance of these two forces determines the aggregate structures. Analysis was followed by the molecular geometrical structure. These findings may have implications for the development of drug delivery systems for cancer treatments, as well as cosmetic and food formulations.

  3. Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

    Science.gov (United States)

    Kanada, Masamitsu; Bachmann, Michael H; Contag, Christopher H

    2016-02-01

    Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Seminal vesicle cystadenoma: a rare clinical perspective.

    Science.gov (United States)

    Lorber, Gideon; Pizov, Galina; Gofrit, Ofer N; Pode, Dov

    2011-08-01

    A 52-yr-old man presented with severe obstructive urinary symptoms. Ten years earlier, a digital rectal examination disclosed a small mass above the prostate, and a computed tomography (CT) scan showed a 3.5-cm cystic tumor of the right seminal vesicle. He had been followed conservatively elsewhere. Reevaluation of the mass with a CT scan and magnetic resonance imaging showed that the mass had grown to a maximal diameter of 14 cm. A transabdominal needle biopsy revealed benign fibromuscular tissue. The tumor was then resected by an open transvesical approach. Pathology was consistent with a benign seminal vesicle cystadenoma. The natural history, pathology, and surgical approach are described. Copyright © 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  5. Docking of secretory vesicles is syntaxin dependent.

    Directory of Open Access Journals (Sweden)

    Heidi de Wit

    Full Text Available Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones.

  6. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    Science.gov (United States)

    Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (inflammation in immune cells. © 2017 American Heart Association, Inc.

  7. A readily retrievable pool of synaptic vesicles

    OpenAIRE

    Hua, Y; Sinha, R.; Thiel, C.; Schmidt, R.; Hueve, J.; Martens, H.; Hell, S.; Egner, A.; Klingauf, J.

    2011-01-01

    Abstract Although clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism of synaptic vesicle (SV) recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support a first wave of fast CME. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of SV proteins in rat hippocampal neurons using a novel probe. Applying...

  8. Endothelial microparticles: Sophisticated vesicles modulating vascular function

    Science.gov (United States)

    Curtis, Anne M; Edelberg, Jay; Jonas, Rebecca; Rogers, Wade T; Moore, Jonni S; Syed, Wajihuddin; Mohler, Emile R

    2015-01-01

    Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation. PMID:23892447

  9. Routes and mechanisms of extracellular vesicle uptake

    Directory of Open Access Journals (Sweden)

    Laura Ann Mulcahy

    2014-08-01

    Full Text Available Extracellular vesicles (EVs are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.

  10. RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone

    Science.gov (United States)

    Han, Yunyun; Kaeser, Pascal S.; Südhof, Thomas C.; Schneggenburger, Ralf

    2012-01-01

    At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles. The mechanisms that enrich Ca2+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knock-out approach at a presynaptically accessible CNS synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca2+ channel density, revealing a new role of RIM proteins in Ca2+ channel targeting. Removal of RIMs also reduced the readily-releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca2+ channel - vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release: enabling a high presynaptic Ca2+ channel density, and vesicle docking at the active zone. PMID:21262468

  11. A Time-Resolved Study on Nanodisc-to-Vesicle Transformation

    Science.gov (United States)

    Nieh, Mu-Ping; Mahabir, Suanne; Wan, Wan Kei; Kastaras, John

    2011-03-01

    Structural phase diagram of a phospholipid mixture composed of dimyristoyl phosphatidylcholine (DMPC), dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylglycerol (DMPG) contains many rich morphologies, e.g., nanodiscs also known as ``bicelles'', bilayered ribbons, unilamellar vesicles (ULVs), multi-lamellar vesicles (MLVs) and perforated lamellae. In this report, we will present time-resolved small angle neutron scattering and dynamic light scattering measurements of the structural transformation from nanodiscs to ULVs as a function of temperature, lipid concentration and charge density. The result will reveal the growth rate of nanodiscs and all the intermediate structures along the transformation process. Through the understanding of the kinetic pathway, the size and polydispersity of the self-assembled nano-size ULVs can be well-controlled. These ULVs can be used as a carrier for therapeutics or imaging probes.

  12. Cholesterol affects the interaction between an ionic liquid and phospholipid vesicles. A study by differential scanning calorimetry and nanoplasmonic sensing.

    Science.gov (United States)

    Russo, Giacomo; Witos, Joanna; Rantamäki, Antti H; Wiedmer, Susanne K

    2017-12-01

    The present work aims at studying the interactions between cholesterol-rich phosphatidylcholine-based lipid vesicles and trioctylmethylphosphonium acetate ([P 8881 ][OAc]), a biomass dissolving ionic liquid (IL). The effect of cholesterol was assayed by using differential scanning calorimetry (DSC) and nanoplasmonic sensing (NPS) measurement techniques. Cholesterol-enriched dipalmitoyl-phosphatidylcholine vesicles were exposed to different concentrations of the IL, and the derived membrane perturbation was monitored by DSC. The calorimetric data could suggest that the binding and infiltration of the IL are delayed in the vesicles containing cholesterol. To clarify our findings, NPS was applied to quantitatively follow the resistance of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine incorporating 0, 10, and 50mol% of cholesterol toward the IL exposure over time. The membrane perturbation induced by different concentrations of IL was found to be a concentration dependent process on cholesterol-free lipid vesicles. Moreover, our results showed that lipid depletion in cholesterol-enriched lipid vesicles is inversely proportional to the increasing amount of cholesterol in the vesicles. These findings support that cholesterol-rich lipid bilayers are less susceptible toward membrane disrupting agents as compared to membranes that do not incorporate any sterols. This probably occurs because cholesterol tightens the phospholipid acyl chain packing of the plasma membranes, increasing their resistance and reducing their permeability. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Titration of Syntaxin1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.

    Science.gov (United States)

    Arancillo, Marife; Min, Sang-Won; Gerber, Stefan; Münster-Wandowski, Agnieszka; Wu, Yuan-Ju; Herman, Melissa; Trimbuch, Thorsten; Rah, Jong-Cheol; Ahnert-Hilger, Gudrun; Riedel, Dietmar; Südhof, Thomas C; Rosenmund, Christian

    2013-10-16

    Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.

  14. A novel method for the isolation of extracellular vesicles and RNA from urine.

    Science.gov (United States)

    Markowska, Anna; Pendergrast, R Scott; Pendergrast, J Stephen; Pendergrast, P Shannon

    2017-01-01

    The discovery of urinary extracellular biomarkers has been impeded by the lack of efficient methods for the isolation of extracellular vesicles (EVs: exosomes and microvesicles) and extracellular nucleic acid (RNA and DNA) from urine. Ultracentrifugation (UC), considered the gold standard for vesicle isolation from many biofluids, is efficacious but laborious, and, like most commercially available methods, is unable to isolate enough material from small volumes for protein or RNA-based biomarker discovery. We have developed a novel precipitation method for the isolation of EVs and nucleic acids from urine. The method, which is now commercially available, takes less than 30 min and does not require polyethylene glycol. Transmission electron microscopy and Nanosight particle analysis confirm that the method isolates intact vesicles with a similar size, shape, and number to UC. Immunoblot analysis of preparations made from a variety of urine samples demonstrates that the method isolates multiple vesicle protein markers more efficiently than other commercial kits, especially from more diluted samples. Bioanalyzer, quantitative reverse transcription polymerase chain reaction, and array analysis show that the method is extremely efficient at the isolation of extracellular miRNAs. The Ymir Genomics EV and Extracellular RNA Isolation Kits offer an efficient and rapid alternative to UC and other commercial kits.

  15. A novel method for the isolation of extracellular vesicles and RNA from urine

    Directory of Open Access Journals (Sweden)

    Anna Markowska

    2017-06-01

    Full Text Available The discovery of urinary extracellular biomarkers has been impeded by the lack of efficient methods for the isolation of extracellular vesicles (EVs: exosomes and microvesicles and extracellular nucleic acid (RNA and DNA from urine. Ultracentrifugation (UC, considered the gold standard for vesicle isolation from many biofluids, is efficacious but laborious, and, like most commercially available methods, is unable to isolate enough material from small volumes for protein or RNA-based biomarker discovery. We have developed a novel precipitation method for the isolation of EVs and nucleic acids from urine. The method, which is now commercially available, takes less than 30 min and does not require polyethylene glycol. Transmission electron microscopy and Nanosight particle analysis confirm that the method isolates intact vesicles with a similar size, shape, and number to UC. Immunoblot analysis of preparations made from a variety of urine samples demonstrates that the method isolates multiple vesicle protein markers more efficiently than other commercial kits, especially from more diluted samples. Bioanalyzer, quantitative reverse transcription polymerase chain reaction, and array analysis show that the method is extremely efficient at the isolation of extracellular miRNAs. The Ymir Genomics EV and Extracellular RNA Isolation Kits offer an efficient and rapid alternative to UC and other commercial kits.

  16. Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes

    Science.gov (United States)

    Pellett, Patrina A; Dietrich, Felix; Bewersdorf, Jörg; Rothman, James E; Lavieu, Grégory

    2013-01-01

    A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI: http://dx.doi.org/10.7554/eLife.01296.001 PMID:24137546

  17. Small-Angle Neutron Scattering and Spontaneous Formation of Unilamellar Vesicles: Potential Vehicles for Drug Delivery

    Science.gov (United States)

    Katsaras, John

    2004-03-01

    Unilamellar vesicles (ULVs) are single-bilayer shells with radii commonly between 10 and 100 nm, and are widely used as model membranes, drug delivery systems, microreactors and substrates for a variety of enzymes and proteins. A common method of making ULVs is the extrusion of multilamellar vesicles (MLVs) through synthetic membranes of known pore size. These extruded ULVs are invariably unstable and in due time, revert back to MLVs. Over the years there have been reports of the spontaneous formation of stable ULVs in surfactant, lipid, and lipid/detergent mixtures. These ULVs have sometimes been shown to be monodisperse and their radii were found, almost without exception, to vary with concentration. We have carried-out small-angle neutron scattering (SANS) experiments on a biomimetic system composed of the phospholipids dimyristoyl and dihexanoyl phosphorylcholine (DMPC and DHPC, respectively). Doping DMPC/DHPC multilamellar vesicles with either the negatively charged lipid dimyristoyl phosphorylglycerol (DMPG, net charge -1) or the divalent cation, calcium (Ca2+) leads to the spontaneous formation of monodisperse unilamellar vesicles whose radii are concentration independent, in contrast to previous experimental observations.

  18. Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

    Science.gov (United States)

    Osteikoetxea, Xabier; Németh, Andrea; Sódar, Barbara W; Vukman, Krisztina V; Buzás, Edit Irén

    2016-06-01

    In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer-enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  19. Syncytiotrophoblast Extracellular Vesicles from Pre-Eclampsia Placentas Differentially Affect Platelet Function.

    Directory of Open Access Journals (Sweden)

    Dionne S Tannetta

    Full Text Available Pre-eclampsia (PE complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition.

  20. Syncytiotrophoblast Extracellular Vesicles from Pre-Eclampsia Placentas Differentially Affect Platelet Function.

    Science.gov (United States)

    Tannetta, Dionne S; Hunt, Kathryn; Jones, Chris I; Davidson, Naomi; Coxon, Carmen H; Ferguson, David; Redman, Christopher W; Gibbins, Jonathan M; Sargent, Ian L; Tucker, Katherine L

    2015-01-01

    Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition.

  1. Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

    DEFF Research Database (Denmark)

    Vind-Kezunovic, Dina; Helix Nielsen, Claus; Wojewodzka, Urszula

    2008-01-01

    on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total 'L-o/L-d' boundary line tension arising from the coalescence of smaller L-d-like domains makes it energetically favourable for L-d-like domains to bend from flat mu m-sized surfaces...... to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events....

  2. Matrix metalloproteinase-10 effectively reduces infarct size in experimental stroke by enhancing fibrinolysis via a thrombin-activatable fibrinolysis inhibitor-mediated mechanism.

    Science.gov (United States)

    Orbe, J; Barrenetxe, J; Rodriguez, J A; Vivien, D; Orset, C; Parks, W C; Birkland, T P; Serrano, R; Purroy, A; Martinez de Lizarrondo, S; Angles-Cano, E; Páramo, J A

    2011-12-20

    The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution and extracellular matrix proteolysis. The plasminogen/plasmin system activates MMPs, and some MMPs have been involved in the dissolution of fibrin by targeting fibrin(ogen) directly or by collaborating with plasmin. MMP-10 has been implicated in inflammatory/thrombotic processes and vascular integrity, but whether MMP-10 could have a profibrinolytic effect and represent a promising thrombolytic agent is unknown. The effect of MMP-10 on fibrinolysis was studied in vitro and in vivo, in MMP-10-null mice (Mmp10(-/-)), with the use of 2 different murine models of arterial thrombosis: laser-induced carotid injury and ischemic stroke. In vitro, we showed that MMP-10 was capable of enhancing tissue plasminogen activator-induced fibrinolysis via a thrombin-activatable fibrinolysis inhibitor inactivation-mediated mechanism. In vivo, delayed fibrinolysis observed after photochemical carotid injury in Mmp10(-/-) mice was reversed by active recombinant human MMP-10. In a thrombin-induced stroke model, the reperfusion and the infarct size in sham or tissue plasminogen activator-treated animals were severely impaired in Mmp10(-/-) mice. In this model, administration of active MMP-10 to wild-type animals significantly reduced blood reperfusion time and infarct size to the same extent as tissue plasminogen activator and was associated with shorter bleeding time and no intracranial hemorrhage. This effect was not observed in thrombin-activatable fibrinolysis inhibitor-deficient mice, suggesting thrombin-activatable fibrinolysis inhibitor inactivation as one of the mechanisms involved in the MMP-10 profibrinolytic effect. A novel profibrinolytic role for MMP-10 in experimental ischemic stroke is described, opening new pathways for innovative fibrinolytic strategies in arterial thrombosis.

  3. Co-administration of the flavanol (-)-epicatechin with doxycycline synergistically reduces infarct size in a model of ischemia reperfusion injury by inhibition of mitochondrial swelling.

    Science.gov (United States)

    Ortiz-Vilchis, Pilar; Yamazaki, Katrina Go; Rubio-Gayosso, Ivan; Ramirez-Sanchez, Israel; Calzada, Claudia; Romero-Perez, Diego; Ortiz, Alicia; Meaney, Eduardo; Taub, Pam; Villarreal, Francisco; Ceballos, Guillermo

    2014-12-05

    (-)-Epicatechin (EPI) is cardioprotective in the setting of ischemia/reperfusion (IR) injury and doxycycline (DOX) is known to preserve cardiac structure/function after myocardial infarction (MI). The main objective of this study was to examine the effects of EPI and DOX co-administration on MI size after IR injury and to determine if cardioprotection may involve the mitigation of mitochondrial swelling. For this purpose, a rat model of IR was used. Animals were subjected to a temporary 45 min occlusion of the left anterior descending coronary artery. Treatment consisted of a single or double dose of EPI (10 mg/kg) combined with DOX (5 mg/kg). The first dose was given 15 min prior to reperfusion and the second 12 h post-MI. The effects of EPI +/- DOX on mitochondrial swelling (i.e. mPTP opening) were determined using isolated mitochondria exposed to calcium overload and data examined using isobolographic analysis. To ascertain for the specificity of EPI effects on mitochondrial swelling other flavonoids were also evaluated. Single dose treatment reduced MI size by ~46% at 48 h and 44% at three weeks. Double dosing evidenced a synergistic, 82% reduction at 3 weeks. EPI plus DOX also inhibited mitochondrial swelling in a synergic manner thus, possibly accounting for the cardioprotective effects whereas limited efficacy was observed with the other flavonoids. Given the apparent lack of toxicity in humans, the combination of EPI and DOX may have clinical potential for the treatment of myocardial IR injury. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Secretable Small RNAs via Outer Membrane Vesicles in Periodontal Pathogens.

    Science.gov (United States)

    Choi, J-W; Kim, S-C; Hong, S-H; Lee, H-J

    2017-04-01

    MicroRNAs (miRNAs) have been shown to be major regulators of eukaryotic gene expression. However, bacterial RNAs comparable in size to eukaryotic miRNAs (18-22 nucleotides) have received little attention. Recently, a novel class of small RNAs similar in size to miRNAs (miRNA-size, small RNAs or msRNAs) have also been found in several bacteria. Like miRNAs, msRNAs are approximately 15 to 25 nucleotides in length, and their precursors are predicted to form a hairpin loop secondary structure. Here, we identified msRNAs in the periodontal pathogens Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola. We examined these msRNAs using a deep sequencing method and characterized dozens of msRNAs through bioinformatic analysis. Highly expressed msRNAs were selected for further validation. The findings suggest that this class of small RNAs is well conserved across the domains of life. Indeed, msRNAs secreted via bacterial outer membrane vesicles (OMVs) were detected. The ability of bacterial OMVs to deliver RNAs into eukaryotic cells was also observed. These msRNAs in OMVs allowed us to identify their potential human immune-related target genes. Furthermore, we found that exogenous msRNAs could suppress expression of certain cytokines in Jurkat T cells. We propose msRNAs may function as novel bacterial signaling molecules that mediate bacteria-to-human interactions. Furthermore, this study may provide fresh insight into bacterial pathogenic mechanisms of periodontal diseases.

  5. Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

    Science.gov (United States)

    Aguilar, Jenny I; Dunn, Matthew; Mingote, Susana; Karam, Caline S; Farino, Zachary J; Sonders, Mark S; Choi, Se Joon; Grygoruk, Anna; Zhang, Yuchao; Cela, Carolina; Choi, Ben Jiwon; Flores, Jorge; Freyberg, Robin J; McCabe, Brian D; Mosharov, Eugene V; Krantz, David E; Javitch, Jonathan A; Sulzer, David; Sames, Dalibor; Rayport, Stephen; Freyberg, Zachary

    2017-08-30

    The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Human mammospheres secrete hormone-regulated active extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Esperanza Gonzalez

    Full Text Available Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression.

  7. Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

    Science.gov (United States)

    Lee, Jaewook; Kim, Si-Hyun; Choi, Dong-Sic; Lee, Jong Seok; Kim, Dae-Kyum; Go, Gyeongyun; Park, Seon-Min; Kim, Si Hyun; Shin, Jeong Hwan; Chang, Chulhun L; Gho, Yong Song

    2015-10-01

    The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. iGUVs: Preparing Giant Unilamellar Vesicles with a Smartphone and Lipids Easily Extracted from Chicken Eggs

    Science.gov (United States)

    Almendro Vedia, Víctor G.; Natale, Paolo; Chen, Su; Monroy, Francisco; Rosilio, Veronique; López-Montero, Ivan

    2017-01-01

    Since the first report of electroformed micrometer-sized liposomes in the 1980s, giant unilamellar vesicles (GUVs) have generated a lot of interest in the biophysical and biochemical communities. However, their penetration rate in high school or at the undergraduate level is still limited because of the requirement of specialized materials for…

  9. Rational synthesis of low-polydispersity block copolymer vesicles in concentrated solution via polymerization-induced self-assembly

    NARCIS (Netherlands)

    Gonzato, Carlo; Semsarilar, Mona; Jones, Elizabeth R.; Li, Feng; Krooshof, Gerard J P; Wyman, Paul; Mykhaylyk, Oleksandr O.; Tuinier, Remco|info:eu-repo/dai/nl/183514262; Armes, Steven P.

    2014-01-01

    Block copolymer self-assembly is normally conducted via post-polymerization processing at high dilution. In the case of block copolymer vesicles (or "polymersomes"), this approach normally leads to relatively broad size distributions, which is problematic for many potential applications. Herein we

  10. Modulation of tissue tropism and biological activity of exosomes and other extracellular vesicles : New nanotools for cancer treatment

    NARCIS (Netherlands)

    Kooijmans, Sander A A|info:eu-repo/dai/nl/413754170; Schiffelers, Raymond M.|info:eu-repo/dai/nl/212909509; Zarovni, Natasa; Vago, Riccardo

    2016-01-01

    Exosomes are naturally secreted nanovesicles that have recently aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. These 30–100 nm sized vesicles are released from the cells into the

  11. DNA-mediated self-assembly of artificial vesicles.

    Science.gov (United States)

    Hadorn, Maik; Eggenberger Hotz, Peter

    2010-03-26

    Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time. In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts. This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and

  12. Cellular phenotype and extracellular vesicles: basic and clinical considerations.

    Science.gov (United States)

    Quesenberry, Peter J; Goldberg, Laura R; Aliotta, Jason M; Dooner, Mark S; Pereira, Mandy G; Wen, Sicheng; Camussi, Giovanni

    2014-07-01

    Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently, vesicles were studied as possible vaccines and, most recently, there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either the lung or liver can enter bone marrow cells (this is a prerequisite) and alter their fate toward that of the originating liver and lung tissue. Lung vesicles interacted with bone marrow cells result in the bone marrow cells expressing surfactants A-D, Clara cell protein, and aquaporin-5 mRNA. In a similar vein, liver-derived vesicles induce albumin mRNA in target marrow cells. The vesicles contain protein, mRNA, microRNA, and noncoding RNA and variably some DNA. This genetic package is delivered to cells and alters the phenotype. Further studies have shown that initially the altered phenotype is due to the transfer of mRNA and a transcriptional modulator, but long-term epigenetic changes are induced through transfer of a transcriptional factor, and the mRNA is rapidly degraded in the cell. Studies on the capacity of vesicles to restore injured tissue have been quite informative. Mesenchymal stem cell-derived vesicles are able to reverse the injury to the damaged liver and kidney. Other studies have shown that mesenchymal stem cell-derived vesicles can reverse radiation toxicity of bone marrow stem cells. Extracellular vesicles offer an intriguing strategy for treating a number of diseases characterized by tissue injury.

  13. Spin State As a Probe of Vesicle Self-Assembly

    OpenAIRE

    Kim, Sanghoon; Bellouard, Christine; Eastoe, Julian; Canilho, Nadia; Rogers, Sarah E; Ihiawakrim, Dris; Ersen, Ovidiu; Pasc, Andreea

    2016-01-01

    A novel system of paramagnetic vesicles was designed using ion pairs of iron-containing surfactants. Unilamellar vesicles (diameter ≈ 200 nm) formed spontaneously and were characterized by cryogenic transmission electron microscopy, nanoparticle tracking analysis, and light and small-angle neutron scattering. Moreover, for the first time, it is shown that magnetization measurements can be used to investigate self-assembly of such functionalized systems, giving information on the vesicle compo...

  14. Spin State As a Probe of Vesicle Self-Assembly.

    Science.gov (United States)

    Kim, Sanghoon; Bellouard, Christine; Eastoe, Julian; Canilho, Nadia; Rogers, Sarah E; Ihiawakrim, Dris; Ersen, Ovidiu; Pasc, Andreea

    2016-03-02

    A novel system of paramagnetic vesicles was designed using ion pairs of iron-containing surfactants. Unilamellar vesicles (diameter ≈ 200 nm) formed spontaneously and were characterized by cryogenic transmission electron microscopy, nanoparticle tracking analysis, and light and small-angle neutron scattering. Moreover, for the first time, it is shown that magnetization measurements can be used to investigate self-assembly of such functionalized systems, giving information on the vesicle compositions and distribution of surfactants between the bilayers and the aqueous bulk.

  15. [Seminal vesicle cystadenoma as the cause of a retrovesical tumor].

    Science.gov (United States)

    Kaminsky, A; Kania, U; Ortloff, P; Sperling, H

    2014-04-01

    Tumors of the seminal vesicle are rare. Malignant tumors are more common than benign tumors. A seminal vesicle cystadenoma is a rarity. We report on a 41-year-old man with the incidental finding of an asymptomatic retrovesical tumor. The tumor, the seminal vesicle, and the abdominal part of the ductus deferens were surgically removed. The operative access is variable and surgical treatment is the method of choice. The patient's prognosis is good and there are no signs of recurrence.

  16. Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

    Directory of Open Access Journals (Sweden)

    Oscarsson Jan

    2008-01-01

    Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. Results The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S and in its spontaneous laboratory variant (D7SS resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm, AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05–0.2 μm were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. Conclusion Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

  17. Floating Escherichia coli by expressing cyanobacterial gas vesicle genes

    Science.gov (United States)

    Wang, Tianhe; Kang, Li; Li, Jiaheng; Wu, Wenjie; Zhang, Peiran; Gong, Minghao; Lai, Weihong; Zhang, Chunyan; Chang, Lei; Peng, Yong; Yang, Zhongzhou; Li, Lian; Bao, Yingying; Xu, Haowen; Zhang, Xiaohua; Sui, Zhenghong; Yang, Guanpin; Wang, Xianghong

    2015-02-01

    Gas vesicles are hollow, air-filled polyprotein structures that provide the buoyancy to cells. They are found in a variety of prokaryotes. In this study, we isolated a partial gas vesicle protein gene cluster containing gvpA and gvpC20Ψ from Planktothrix rubescens, and inserted it into an expression vector and expressed it in E. coli. The gas vesicle was developed in bacterial cells, which made bacterial cells to float on medium surface. We also amplified gvpA and gvpC20Ψ separately and synthesized an artificial operon by fusing these two genes with the standardized gene expression controlling elements of E. coli. The artificial operon was expressed in E. coli, forming gas vesicles and floating bacteria cells. Our findings verified that the whole set of genes and the overall structure of gas vesicle gene cluster are not necessary for developing gas vesicles in bacteria cells. Two genes, gvpA and gvpC20Ψ, of the gas vesicle gene cluster are sufficient for synthesizing an artificial operon that can develop gas vesicles in bacteria cells. Our findings provided a wide range of applications including easing the harvest of cultured microalgae and bacteria, as well as enriching and remediating aquatic pollutants by constructing gas vesicles in their cells.

  18. Cholesterol Regulates Multiple Forms of Vesicle Endocytosis at a Mammalian Central Synapse

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2015-01-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. PMID:25893258

  19. Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles.

    Science.gov (United States)

    Bakhti, Mostafa; Winter, Christine; Simons, Mikael

    2011-01-07

    Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis.

  20. Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2015-07-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. © 2015 International Society for Neurochemistry.

  1. Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland.

    Science.gov (United States)

    González, Débora Alejandra; Barbieri van Haaster, Martín Matías; Quinteros Villarruel, Emmanuel; Brandt, Macarena; Benítez, María Belén; Stranieri, Graciela Mabel; Orman, Betina

    2018-01-01

    Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H 4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgADP or MgAMP. Histamine increased the release of vesicles with nucleotidase activity in a concentration dependent manner. JNJ7777120 significantly reduced this effect. Vesicular nucleotidases obtained in the absence or presence of histamine promoted Pi production from ATP, ADP and AMP. The results show a relationship between histamine and the regulation of purinergic signaling, which could be important in the modulation of inflammatory processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes.

    Science.gov (United States)

    Ascenso, Andreia; Raposo, Sara; Batista, Cátia; Cardoso, Pedro; Mendes, Tiago; Praça, Fabíola Garcia; Bentley, Maria Vitória Lopes Badra; Simões, Sandra

    2015-01-01

    Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by "transfersomal method", for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by "ethosomal method". Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes