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Sample records for vesicles bbmv isolated

  1. Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

    International Nuclear Information System (INIS)

    Goldman, M.; Yaari, A.; Moran, A.; Doshnitzki, Z.; Cohen-Luria, R.

    2006-01-01

    Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K i of uranyl was 139±30 μg uranyl/mg protein of BBMV. Uranyl at 140 μg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V max 2.2±0.2 and 0.96±0.16 nmol/mg protein for control and uranyl treated BBMV (P m (1.54±0.33 and 1.54±0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V max is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V max was reduced from 247±13 pmol/mg protein in control BBMV to 119±3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex. (orig.)

  2. Methods to isolate extracellular vesicles for diagnosis

    Science.gov (United States)

    Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

    2017-12-01

    Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

  3. Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics.

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    Julia I Tandberg

    Full Text Available Membrane vesicles (MVs are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89, Norway (NVI 5692 and Canada (NVI 5892, respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium's utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.

  4. Sulfate uptake by crustacean hepatopancreatic brush border membrane vesicles

    International Nuclear Information System (INIS)

    Gerencser, G.A.; Cattey, M.A; Ahearn, G.A.

    1990-01-01

    Purified brush border membrane vesicles (BBMV) were prepared from Atlantic lobster (Homarus americanus) hepatopancreas using differential centrifugation and Mg +2 precipitation techniques. Uptake of 0.1 mM 35 SO 4 -2 was stimulated by pre-loading vesicles with Cl - leading to a transient accumulation of isotope more than twice that at equilibrium. Pre-loading with HCO 3 - or gluconate had no effect on sulfate uptake. No stimulation of 35 SO 4 -2 was observed in the presence of inwardly directed Na + or tetramethylammonium + gradients. Uptake of the divalent anion was strongly stimulated by inwardly directed proton gradients (pH o i ) and markedly inhibited by outwardly directed proton gradients (pH o > pH i ). 35 SO 4 -2 /Cl - exchange was enhanced by imposing a transmembrane inside positive K + diffusion potential and inhibited by a membrane potential of the opposite polarity (K + /valinomycin). Results suggest the presence of a proton-dependent, electrogenic anion antiport mechanism in BBMV isolated from the crustacean hepatopancreas

  5. Methods for extracellular vesicles isolation in a hospital setting

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    Matías eSáenz-Cuesta

    2015-02-01

    Full Text Available The research in extracellular vesicles (EVs has been rising during the last decade. However, there is no clear consensus on the most accurate protocol to isolate and analyze them. Besides, most of the current protocols are difficult to implement in a hospital setting due to being very time consuming or to requirements of specific infrastructure. Thus, our aim is to compare five different protocols (comprising two different medium-speed differential centrifugation protocols; commercially polymeric precipitation -exoquick-; acid precipitation; and ultracentrifugation for blood and urine samples to determine the most suitable one for the isolation of EVs. Nanoparticle tracking analysis, flow cytometry, western blot, electronic microscopy and spectrophotometry were used to characterize basic aspects of EVs such us concentration, size distribution, cell-origin and transmembrane markers and RNA concentration. The highest EV concentrations were obtained using the exoquick protocol, followed by both differential centrifugation protocols, while the ultracentrifugation and acid-precipitation protocols yielded considerably lower EV concentrations. The five protocols isolated EVs of similar characteristics regarding markers and RNA concentration however standard protocol recovered only small EVs. EV isolated with exoquick presented difficult to be analyzed with western blot. The RNA concentrations obtained from urine-derived EVs were similar to those obtained from blood-derived ones, despite the urine EV concentration being 10 to 20 times lower. We consider that a medium-speed differential centrifugation could be suitable to be applied in a hospital setting due to require the simplest infrastructure and recover higher concentration of EV than standard protocol. A workflow from sampling to characterization of EVs is proposed.

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

    DEFF Research Database (Denmark)

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

    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...... is observed all over the gradient. The variety of the membrane vesicles is currently being investigated further by several means. Summary/conclusion: A new procedure for easy and gentle isolation of bovine milk membrane vesicles encompassing ultracentrifugation and size-exclusion chromatography has been...... established. The resulting vesicle isolate exhibits the general membrane vesicle characteristics and provides an appropriate start material from which the variety of milk vesicles can be investigated...

  7. 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.

  8. Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.

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    Merchant, Michael L; Rood, Ilse M; Deegens, Jeroen K J; Klein, Jon B

    2017-12-01

    Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

  9. 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

    as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk...... 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...

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

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    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.

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

    Science.gov (United States)

    Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

    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 from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

  12. 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

    -marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides...... 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...... from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV...

  13. Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures

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    Marijke I. Zonneveld

    2014-08-01

    Full Text Available Extracellular vesicles (EV in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at −80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system.

  14. Comparative miRNA Analysis of Urine Extracellular Vesicles Isolated through Five Different Methods

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    Felix Royo

    2016-12-01

    Full Text Available Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

  15. Comparative proteomic analysis of extracellular vesicles isolated by acoustic trapping or differential centrifugation

    NARCIS (Netherlands)

    Rezeli, Melinda; Gidlöf, Olof; Evander, Mikael; Bryl-Górecka, Paulina; Sathanoori, Ramasri; Gilje, Patrik; Pawlowski, Krzysztof; Horvatovich, Péter; Erlinge, David; Marko-Varga, György; Laurell, Thomas

    2016-01-01

    Extracellular vesicles (ECVs), including microparticles (MPs) and exosomes, are submicron membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared

  16. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

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    Kenneth W. Witwer

    2013-05-01

    Full Text Available The emergence of publications on extracellular RNA (exRNA and extracellular vesicles (EV has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

  17. Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa

    International Nuclear Information System (INIS)

    Hall, K.; Perez, G.; Anderson, D.; Gutierrez, C.; Munson, K.; Hersey, S.J.; Kaplan, J.H.; Sachs, G.

    1990-01-01

    The glycosylation of H+K(+)-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K(+)-ATPase beta-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles

  18. Isolation and Characterization of Serum Extracellular Vesicles (EVs from Atlantic Salmon Infected with Piscirickettsia Salmonis

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    Leidy Lagos

    2017-12-01

    Full Text Available Secretion of extracellular vesicles (EVs is a common feature of both eukaryotic and prokaryotic cells. Isolated EVs have been shown to contain different types of molecules, including proteins and nucleic acids, and are reported to be key players in intercellular communication. Little is known, however, of EV secretion in fish, or the effect of infection on EV release and content. In the present study, EVs were isolated from the serum of healthy and Piscirickettsia salmonis infected Atlantic salmon in order to evaluate the effect of infection on EV secretion. P. salmonis is facultative intracellular bacterium that causes a systemic infection disease in farmed salmonids. EVs isolated from both infected and non-infected fish had an average diameter of 230–300 nm, as confirmed by transmission electron microscopy, nanoparticle tracking, and flow cytometry. Mass spectrometry identified 180 proteins in serum EVs from both groups of fish. Interestingly, 35 unique proteins were identified in serum EVs isolated from the fish infected with P. salmonis. These unique proteins included proteasomes subunits, granulins, and major histocompatibility class I and II. Our results suggest that EV release could be part of a mechanism in which host stimulatory molecules are released from infected cells to promote an immune response.

  19. Zinc uptake in swine intestinal brush border membrane vesicles using a 65Zn/69mZn duel isotope experiment

    International Nuclear Information System (INIS)

    Huntington, C.E.; Morris, J.S.; Veum, T.L.

    2008-01-01

    Supplemental zinc as ZnO is routinely added to nursery pig diets at 15 to 20 times the nutritional requirement to alleviate physiological stress and enhance performance. The mechanism by which Zn accomplishes this function is unknown, however, high concentrations of Zn are excreted in the environment at undesirable levels. To study Zn uptake, we developed a multi-stage digestion model followed by exposure to swine intestinal brush border membrane vesicles (BBMV). We report on the feasibility of using a duel label ( 65 Zn and 69m Zn) to simultaneously quantify the competitive uptake of Zn from co-existing zinc supplements using our BBMV model. (author)

  20. Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression.

    Science.gov (United States)

    Willms, Eduard; Cabañas, Carlos; Mäger, Imre; Wood, Matthew J A; Vader, Pieter

    2018-01-01

    Cells release membrane enclosed nano-sized vesicles termed extracellular vesicles (EVs) that function as mediators of intercellular communication by transferring biological information between cells. Tumor-derived EVs have emerged as important mediators in cancer development and progression, mainly through transfer of their bioactive content which can include oncoproteins, oncogenes, chemokine receptors, as well as soluble factors, transcripts of proteins and miRNAs involved in angiogenesis or inflammation. This transfer has been shown to influence the metastatic behavior of primary tumors. Moreover, tumor-derived EVs have been shown to influence distant cellular niches, establishing favorable microenvironments that support growth of disseminated cancer cells upon their arrival at these pre-metastatic niches. It is generally accepted that cells release a number of major EV populations with distinct biophysical properties and biological functions. Exosomes, microvesicles, and apoptotic bodies are EV populations most widely studied and characterized. They are discriminated based primarily on their intracellular origin. However, increasing evidence suggests that even within these EV populations various subpopulations may exist. This heterogeneity introduces an extra level of complexity in the study of EV biology and function. For example, EV subpopulations could have unique roles in the intricate biological processes underlying cancer biology. Here, we discuss current knowledge regarding the role of subpopulations of EVs in cancer development and progression and highlight the relevance of EV heterogeneity. The position of tetraspanins and integrins therein will be highlighted. Since addressing EV heterogeneity has become essential for the EV field, current and novel techniques for isolating EV subpopulations will also be discussed. Further dissection of EV heterogeneity will advance our understanding of the critical roles of EVs in health and disease.

  1. Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression

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    Eduard Willms

    2018-04-01

    Full Text Available Cells release membrane enclosed nano-sized vesicles termed extracellular vesicles (EVs that function as mediators of intercellular communication by transferring biological information between cells. Tumor-derived EVs have emerged as important mediators in cancer development and progression, mainly through transfer of their bioactive content which can include oncoproteins, oncogenes, chemokine receptors, as well as soluble factors, transcripts of proteins and miRNAs involved in angiogenesis or inflammation. This transfer has been shown to influence the metastatic behavior of primary tumors. Moreover, tumor-derived EVs have been shown to influence distant cellular niches, establishing favorable microenvironments that support growth of disseminated cancer cells upon their arrival at these pre-metastatic niches. It is generally accepted that cells release a number of major EV populations with distinct biophysical properties and biological functions. Exosomes, microvesicles, and apoptotic bodies are EV populations most widely studied and characterized. They are discriminated based primarily on their intracellular origin. However, increasing evidence suggests that even within these EV populations various subpopulations may exist. This heterogeneity introduces an extra level of complexity in the study of EV biology and function. For example, EV subpopulations could have unique roles in the intricate biological processes underlying cancer biology. Here, we discuss current knowledge regarding the role of subpopulations of EVs in cancer development and progression and highlight the relevance of EV heterogeneity. The position of tetraspanins and integrins therein will be highlighted. Since addressing EV heterogeneity has become essential for the EV field, current and novel techniques for isolating EV subpopulations will also be discussed. Further dissection of EV heterogeneity will advance our understanding of the critical roles of EVs in health and

  2. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

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    Kazuya Iwai

    2016-05-01

    Full Text Available Diagnostic methods that focus on the extracellular vesicles (EVs present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA and microRNA (miRNA, which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm and higher density (1.11 g/ml than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively. Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

  3. Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

    NARCIS (Netherlands)

    Merchant, M.L.; Rood, I.M.; Deegens, J.K.J.; Klein, J.B.

    2017-01-01

    Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies.

  4. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method

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    Enderle, Daniel; Spiel, Alexandra; Coticchia, Christine M.; Berghoff, Emily; Mueller, Romy; Schlumpberger, Martin; Sprenger-Haussels, Markus; Shaffer, Jonathan M.; Lader, Eric; Skog, Johan; Noerholm, Mikkel

    2015-01-01

    Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as “exoRNeasy Serum/Plasma Maxi Kit”. PMID:26317354

  6. Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

    International Nuclear Information System (INIS)

    Kasai, M.; Muto, S.

    1990-01-01

    Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment. 45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(Km(Ca2+) = 0.4 microM) and ATP(Km(ATP) = 3.9 microM), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl- or NO3-. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl- was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanide m-chlorophenylhydrazone (CCCP) and VO4(3-) which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl(-)-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl(-)-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl(-)-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves

  7. Isolation, Characterization and Biological Properties of Membrane Vesicles Produced by the Swine Pathogen Streptococcus suis.

    Directory of Open Access Journals (Sweden)

    Bruno Haas

    Full Text Available Streptococcus suis, more particularly serotype 2, is a major swine pathogen and an emerging zoonotic agent worldwide that mainly causes meningitis, septicemia, endocarditis, and pneumonia. Although several potential virulence factors produced by S. suis have been identified in the last decade, the pathogenesis of S. suis infections is still not fully understood. In the present study, we showed that S. suis produces membrane vesicles (MVs that range in diameter from 13 to 130 nm and that appear to be coated by capsular material. A proteomic analysis of the MVs revealed that they contain 46 proteins, 9 of which are considered as proven or suspected virulence factors. Biological assays confirmed that S. suis MVs possess active subtilisin-like protease (SspA and DNase (SsnA. S. suis MVs degraded neutrophil extracellular traps, a property that may contribute to the ability of the bacterium to escape the host defense response. MVs also activated the nuclear factor-kappa B (NF-κB signaling pathway in both monocytes and macrophages, inducing the secretion of pro-inflammatory cytokines, which may in turn contribute to increase the permeability of the blood brain barrier. The present study brought evidence that S. suis MVs may play a role as a virulence factor in the pathogenesis of S. suis infections, and given their composition be an excellent candidate for vaccine development.

  8. Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine.

    Science.gov (United States)

    Pellegrini, Kathryn L; Patil, Dattatraya; Douglas, Kristen J S; Lee, Grace; Wehrmeyer, Kathryn; Torlak, Mersiha; Clark, Jeremy; Cooper, Colin S; Moreno, Carlos S; Sanda, Martin G

    2017-06-01

    The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs. Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing. RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG. Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection. © 2017 Wiley Periodicals, Inc.

  9. Metabolism of polyunsaturated (n-3) fatty acids by monkey seminal vesicles: isolation and biosynthesis of omega-3 epoxides.

    Science.gov (United States)

    Oliw, E H; Sprecher, H W

    1991-11-27

    Monooxygenases of monkey seminal vesicles can metabolize arachidonic acid (20:4(n-6)) by w3-hydroxylation to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and eicosapentaenoic acid (20:5(n-3)) to 17,18-dihydroxyeicosatetraenoic acid (Oliw, E.H. (1989) J. Biol. Chem. 264, 17845-17853). The present study aimed to further characterize the oxygenation of (n-3) polyunsaturated fatty acids. 14C-Labelled 22:6(n-3), 20:5(n-3), 20:4-(n-3) and 18:3(n-3) were incubated with microsomes of seminal vesicles of the cynomolgus monkey, NADPH and a cyclooxygenase inhibitor, diclofenac, and the main metabolites were identified by capillary gas chromatography-mass spectrometry. 22:6(n-3) was slowly metabolized to 19,20-dihydroxy-4,7,10,13,16-docosapentaenoic acid, while 20:5(n-3), 20:4(n-3) and 18:3(n-3) were metabolized more efficiently to the corresponding w4,w3-diols. The w3 epoxides, which were obtained from 20:5(n-3) and 18:3(n-3), were isolated in the presence of an epoxide hydrolase inhibitor, 1(2)epoxy-3,3,3-trichloropropane, and the geometry of the epoxides was determined to be 17S, 18R and 15S, 16R, respectively. While 20:5(n-3) was metabolized almost exclusively to the epoxide and diol pair of metabolites, 18:3(n-3) was metabolized not only to the w3 epoxide and the corresponding diol, but also to the w2 alcohol, 17(R)-hydroxy-9,12,15-octadecatrienoic acid. 22:6(n-3) and 5,8,11,14-eicosatetraynoic acid inhibited the biosynthesis of 18(R)-HETE from arachidonic acid (IC50 0.16 and 0.14 mM, respectively). In comparison with 20:4 or 18:3(n-3), 18:1(n-9) and 22:5(n-6) appeared to be slowly metabolized by seminal monooxygenases, while 18:2(n-6) was converted to the w3 alcohol and to smaller amounts of the w2 alcohol (4:1). Together, the results indicate that the w3-hydroxylase and w3-epoxygenase enzyme(s) metabolize 20:4(n-6) and 20:5(n-3) almost exclusively to the w3(R) alcohol and the w3(R, S) epoxide, respectively, while longer and shorter fatty acids either are poor

  10. Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells

    NARCIS (Netherlands)

    M. Franquesa (Marcella); M.J. Hoogduijn (Martin); E. Ripoll (Elia); F. Luk (Franka); M. Salih (Mahdi); M.G.H. Betjes (Michiel); J. Torras; C.C. Baan (Carla); J. Grinyo (Josep); A. Merino (Ana)

    2014-01-01

    textabstractThe research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of

  11. Properties of the mitochondrial carrier of adenine-nucleotide after purification. Study of the transport protein under isolated form and reincorporated form in phospho-lipidic vesicles

    International Nuclear Information System (INIS)

    Brandolin, Gerard

    1983-01-01

    The first part of this research thesis addresses the reconstitution of the ADP/ATP transport by incorporation of the specific carrier, isolated in presence of detergent, in phospholipids vesicles. Fundamental properties of the reconstituted transport are identical to that of transport in mitochondria, notably as far as the exchange stoichiometry, the turn over and the transport Km are concerned, as well as the asymmetric orientation of the carrier in the membrane. The second part of this research addresses the study of interactions of specific ligands with the ADP/ATP transport protein in presence of detergent. The study of the variations of the intrinsic fluorescence of the isolated ADP/ATP carrier highlights conformational changes exclusively induced by the presence of transportable nucleotides which are modulated in a different manner by carboxy-atractyloside or bongkrekic acid. Moreover, by using the isolated protein, a detailed analysis of binding parameters of fluorescent analogues of ATP is reported [fr

  12. Vesicle electrohydrodynamics.

    Science.gov (United States)

    Schwalbe, Jonathan T; Vlahovska, Petia M; Miksis, Michael J

    2011-04-01

    A small amplitude perturbation analysis is developed to describe the effect of a uniform electric field on the dynamics of a lipid bilayer vesicle in a simple shear flow. All media are treated as leaky dielectrics and fluid motion is described by the Stokes equations. The instantaneous vesicle shape is obtained by balancing electric, hydrodynamic, bending, and tension stresses exerted on the membrane. We find that in the absence of ambient shear flow, it is possible that an applied stepwise uniform dc electric field could cause the vesicle shape to evolve from oblate to prolate over time if the encapsulated fluid is less conducting than the suspending fluid. For a vesicle in ambient shear flow, the electric field damps the tumbling motion, leading to a stable tank-treading state.

  13. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs

    Directory of Open Access Journals (Sweden)

    Huinan Li

    2017-06-01

    Full Text Available Synaptic vesicles (SVs are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS of Mus musculus (mouse contain small ribonucleic acids (sRNAs; ≤50 nucleotides (Scientific Reports, 5:1–14(14918 Li et al. (2015 [1]. Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively. We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  14. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris

    Directory of Open Access Journals (Sweden)

    Niehaus Karsten

    2008-06-01

    Full Text Available Abstract Background Outer membrane vesicles (OMVs are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. Results We have demonstrated that Xanthomonas campestris pv. campestris (Xcc releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Conclusion Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

  15. Proline transport by brush-border membrane vesicles of lobster antennal glands

    International Nuclear Information System (INIS)

    Behnke, R.D.; Wong, R.K.; Huse, S.M.; Reshkin, S.J.; Ahearn, G.A.

    1990-01-01

    Purified brush-border membrane vesicles (BBMV) of lobster antennal gland labyrinth and bladder were separately formed by a magnesium precipitation technique. L-[3H]proline uptake was stimulated by a transmembrane NaCl gradient [outside (o) greater than inside (i)] to a greater extent in BBMV from labyrinth than those from the bladder. Detailed study of the labyrinth proline-transport processes revealed a specific dependence on NaCl, with negligible stimulatory effects by NaSCN, Na-gluconate, or KCl. A transmembrane proton gradient (o greater than i) was without stimulatory effect on proline transport. A transmembrane potential difference alone, in the presence of equilibrated NaCl and L-[3H]proline, led to net influx of the labeled amino acid, suggesting that the uptake process was electrogenic and capable of bringing about the net transfer of positive charge to the vesicle interior. Although a transmembrane Na gradient alone, in the presence of equilibrated Cl and L-[3H]proline, was able to bring about the net influx of the amino acid, a transmembrane Cl gradient alone under Na- and L-[3H]proline-equilibrated conditions was not, suggesting that only the Na gradient could energize the carrier process through cotransport, while the anion served an essential activating role. Proline influx by these vesicles occurred by the combination of at least one saturable Michaelis-Menten carrier system (apparent Kt = 0.37 mM; apparent JM = 1.19 nmol.mg protein-1.10 s-1) and apparent diffusion (P = 0.33 nmol.mg protein-1.10 s-1.mM-1). Static head analysis of the transport process suggested a cotransport stoichiometry of 2 Na:1 proline with essential activation by Cl ion

  16. Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

    Science.gov (United States)

    Li, Y M; Chrambach, A

    2001-11-01

    %. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.

  17. Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

    Science.gov (United States)

    Čujová, Sabína; Bednárová, Lucie; Slaninová, Jiřina; Straka, Jakub; Čeřovský, Václav

    2014-11-01

    The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic β-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  18. Are calcifying matrix vesicles in atherosclerotic lesions of cellular origin?

    Science.gov (United States)

    Bobryshev, Yuri V; Killingsworth, Murray C; Huynh, Thuan G; Lord, Reginald S A; Grabs, Anthony J; Valenzuela, Stella M

    2007-03-01

    Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.

  19. 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.

  20. Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells

    Czech Academy of Sciences Publication Activity Database

    Čujová, Sabína; Bednárová, Lucie; Slaninová, Jiřina; Straka, J.; Čeřovský, Václav

    2014-01-01

    Roč. 20, č. 11 (2014), s. 885-895 ISSN 1075-2617 Institutional support: RVO:61388963 Keywords : antimicrobial peptides * wild-bee venom * CD spectroscopy * large unilamellar vesicles * membrane permeabilization * electron microscopy Subject RIV: CE - Biochemistry Impact factor: 1.546, year: 2014

  1. Free amino acids in synaptic vesicles isolated from the cerebellum and cerebral hemispheres of control and neonatally X-irradiated rats

    International Nuclear Information System (INIS)

    Valcana, T.; Hudson, D.B.; Timiras, P.S.

    1984-01-01

    X-irradiation of the rat brain (1000R, at two days of age), suppresses the normal age-related increase in the weight of the cerebellum and cerebral hemispheres and influences amino acid levels. The decrease in glutamic acid concentration, particularly in the cerebellum, supports the previously advanced proposition that this amino acid may be associated with or may be the transmitter of, the rat cerebellar granule cells. Subfractionation of the cerebellar tissue reveals that the decrease in the glutamic acid level consequent to the loss of granule cells, is reflected in the cytoplasmic fraction but not in the synaptic vesicle subfraction, where glutamic acid was increased. The reduced weight gain in the cerebral hemispheres after irradiation, is accompanied by a significant decrease of aspartate in the cytoplasmic fraction, changes which suggest that a specific cell type, with aspartic acid as its neurotransmitter (possibly in the hippocampus), may also be radiosensitive in the early postnatal period. In contrast, in the synaptic vesicle fraction from cerebral hemispheres, all free amino acids, with the exception of glutamine, increased significantly. Overall, the changes in free amino acid concentration induced by X-irradiation in the cytoplasmic fraction in both brain regions studied are opposite to those found in the synaptic vesicle fraction and although they may indicate changes in specific cell populations, as proposed above, they could also reflect changes in cellular compartmentalization and metabolism or changes in the relative axonal arborization of the affected regions

  2. 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.

  3. 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....

  4. Seminal vesicle cycts

    International Nuclear Information System (INIS)

    Alpern, M.B.; Dorfman, R.E.; Gross, B.H.; Gottlieb, C.A.; Sandler, M.A.

    1990-01-01

    PURPOSE: Adult polycystic kidney disease (APKCD), an autosomal dominant disorder, causes cyst formation in the kidney, liver, pancreas, esophagus, ovaries, uterus, and brain. This paper describes four APKCD patients with CT evidence of seminal vesicle cysts (SVCs). Four patients (aged 45-65 years) underwent abdominal/pelvic CT with oral and intravenous contrast material. Three were evaluated for possible renal transplantation and one for sepsis material. All seminal vesicles contained cystic masses with fluid that measured between 0 and 30 HU. Seminal vesicle thickness was 3-4 cm (normal, 1.5 cm). High-density walls separated the 3-12-mm diameter cysts. All patients demonstrated typical renal stigmata of APKCD. One patient had hepatic cysts, and none had cysts elsewhere. Postmortem examination in one patient confirmed the SVCs

  5. Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Kuehn Meta J

    2009-02-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF. To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. Results Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER marker, TRAPα, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939, an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. Conclusion These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

  6. Vesicle-based rechargeable batteries

    Energy Technology Data Exchange (ETDEWEB)

    Stanish, I.; Singh, A. [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave., S.W., Washington, DC 20375 (United States); Lowy, D.A. [Nova Research, Inc., 1900 Elkin St., Alexandria, VA 22308 (United States); Hung, C.W. [Department of Chemical Engineering, University of Maryland, College Park, MD 20742 (United States)

    2005-05-02

    Vesicle-based rechargeable batteries can be fabricated by mounting polymerized vesicles filled with ferrocyanide or ferricyanide to a conductive surface. The potential can be adjusted by changing the concentration ratio of hydroquinone and benzoquinone bound to the vesicle membranes. These batteries show promise as a means of supplying portable power for future autonomous nanosystems. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

  7. Mechanism of cis- and trans-substrate interactions at the tetraethylammonium/H+ exchanger of rabbit renal brush-border membrane vesicles

    International Nuclear Information System (INIS)

    Wright, S.H.; Wunz, T.M.

    1988-01-01

    The kinetic basis for trans-effects of intravesicular substrates on the uptake of the organic cation, tetraethylammonium (TEA), into rabbit renal brush-border membrane vesicles (BBMV) was studied. Preloading BBMV with 1, 2, or 4 mM TEA stimulated the initial rate of uptake and the total net accumulation of 0.1 mM [ 3 H]TEA. The stimulatory effect of intravesicular TEA on the initial rate of uptake was a saturable function of the trans-TEA concentration, with a half-maximal effect noted at an intravesicular concentration of 0.28 mM. A 1 mM trans-concentration of TEA increased the Jmax of [ 3 H]TEA uptake (from 4.3 to 6.8 nmol.mg-1.min-1) without affecting the apparent Kt. An outwardly directed H+ gradient also increased Jmax (to 10.7 nmol.mg-1.min-1), although the addition of an outwardly directed TEA gradient did not produce further increases in the rate of TEA uptake. External H+ acted as a competitive inhibitor of TEA uptake, and an increase in external [H+] (from 32 nM to 100 nM) produced an increase in the apparent Kt for TEA transport (from 0.12 to 0.26 mM) without affecting the Jmax. The results suggested that TEA and H+ compete for a common site or set of mutually exclusive sites on the cytoplasmic and luminal aspects of TEA/H+ exchanger in the renal brush border, and that these sites have a similar affinity for TEA

  8. Generic sorting of raft lipids into secretory vesicles in yeast

    DEFF Research Database (Denmark)

    Surma, Michal A; Klose, Christian; Klemm, Robin W

    2011-01-01

    Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma...... a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic...

  9. 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.

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

    International Nuclear Information System (INIS)

    Campbell, K.P.

    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 Ca 2+ + Mg 2+ 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 Ca 2+ + Mg 2+ 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 Ca 2+ /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

  11. 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.

  12. 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.

  13. Plane partition vesicles

    International Nuclear Information System (INIS)

    Rensburg, E J Janse van; Ma, J

    2006-01-01

    We examine partitions and their natural three-dimensional generalizations, plane partitions, as models of vesicles undergoing an inflation-deflation transition. The phase diagrams of these models include a critical point corresponding to an inflation-deflation transition, and exhibits multicritical scaling in the vicinity of a multicritical point located elsewhere on the critical curve. We determine the locations of the multicritical points by analysing the generating functions using analytic and numerical means. In addition, we determine the numerical values of the multicritical scaling exponents associated with the multicritical scaling regimes in these models

  14. Vesicles and vesicle fusion: coarse-grained simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2010-01-01

    of vesicles that is crucial for this transport is their ability to fuse to target membranes and release their contents to the distal side. In industry, some personal care products contain vesicles to help transport reagents across the skin, and research on drug formulation shows that packaging active......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...

  15. Calmodulin stimulation of calcium transport in carrot microsomal vesicles

    International Nuclear Information System (INIS)

    Pierce, W.S.; Sze, H.

    1987-01-01

    ATP-dependent 45 Ca 2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca 2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca 2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca 2+ . These results are consistent with the ER being an important site of intracellular Ca 2+ regulation

  16. 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

    , 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...... in vesicle number and size, whereas the fusion competence of generated vesicles was unaffected by the absence of PICK1. Viral rescue experiments demonstrated that long-term re-expression of PICK1 is necessary to restore normal vesicular content and secretion, while short-term overexpression is ineffective...

  17. Molecular characterization of exosome-like vesicles from breast cancer cells

    International Nuclear Information System (INIS)

    Kruger, Stefan; Elmageed, Zakaria Y Abd; Hawke, David H; Wörner, Philipp M; Jansen, David A; Abdel-Mageed, Asim B; Alt, Eckhard U; Izadpanah, Reza

    2014-01-01

    Membrane vesicles released by neoplastic cells into extracellular medium contain potential of carrying arrays of oncogenic molecules including proteins and microRNAs (miRNA). Extracellular (exosome-like) vesicles play a major role in cell-to-cell communication. Thus, the characterization of proteins and miRNAs of exosome-like vesicles is imperative in clarifying intercellular signaling as well as identifying disease markers. Exosome-like vesicles were isolated using gradient centrifugation from MCF-7 and MDA-MB 231 cultures. Proteomic profiling of vesicles using liquid chromatography-mass spectrometry (LC-MS/MS) revealed different protein profiles of exosome-like vesicles derived from MCF-7 cells (MCF-Exo) than those from MDA-MB 231 cells (MDA-Exo). The protein database search has identified 88 proteins in MDA-Exo and 59 proteins from MCF-Exo. Analysis showed that among all, 27 proteins were common between the two exosome-like vesicle types. Additionally, MDA-Exo contains a higher amount of matrix-metalloproteinases, which might be linked to the enhanced metastatic property of MDA-MB 231 cells. In addition, microarray analysis identified several oncogenic miRNA between the two types vesicles. Identification of the oncogenic factors in exosome-like vesicles is important since such vesicles could convey signals to non-malignant cells and could have an implication in tumor progression and metastasis

  18. Removal of Vesicle Structures from Transmission Electron Microscope Images

    DEFF Research Database (Denmark)

    Jensen, Katrine Hommelhoff; Sigworth, Fred; Brandt, Sami Sebastian

    2015-01-01

    In this paper, we address the problem of imaging membrane proteins for single-particle cryo-electron microscopy reconstruction of the isolated protein structure. More precisely, we propose a method for learning and removing the interfering vesicle signals from the micrograph, prior to reconstruct...

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

    International Nuclear Information System (INIS)

    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 topical issue is the dynamics of vesicle formation/breakdown, as the understanding of the transition process will open the way to a deeper understanding of their stability and also allow controlling of the structures formed, by means of their formation processes. Significant progress in the study of the transformation processes has been achieved, in particular by means of time-resolved scattering experiments. (topical review)

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

    KAUST Repository

    Wang, Mingfeng; Zhang, Meng; Siegers, Conrad; Scholes, Gregory D.; Winnik, Mitchell A.

    2009-01-01

    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

  1. Thermally assisted acoustofluidic separation of extracellular vesicles from cells

    Science.gov (United States)

    Mirtaheri, Elnaz; Dolatmoradi, Ata; Pimentel, Krystine; Bhansali, Shekhar; El-Zahab, Bilal

    2018-02-01

    Extracellular vesicles (EVs) have been gaining increasing attention given their role in communicating information between cells. Composition-based isolation of EVs is particularly of high significance as the proteomic and lipidomic characterization of their cargo could provide valuable clues to the role of EVs in mediating the biology of various conditions. This has, however, proved to be challenging as EVs, despite their abundance, are very small and difficult to be differentiated from the other constituents of host media. In addition, currently available methods like ultracentrifugation and filtration are cumbersome and capable of achieving mostly size-based separations. In this work, we demonstrate the possibility of separating submicron EV-like vesicles from cancer cells using a thermally-assisted acoustophoretic device. In a system composed of MCF-7 breast cancer cells spiked with two different types of same-size vesicles, composition-based isolation of vesicles was shown to be realizable through opposite focusing of the system's components at the node and antinodes of the overlaid ultrasonic standing wave. By proper choice of temperature in the microchannel, we were able to achieve separations with purities exceeding 93%. Furthermore, cells recovered from the channel were shown to be viable after the separation.

  2. Extracellular vesicles: Exosomes, microvesicles, and friends

    NARCIS (Netherlands)

    Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

  3. Spontaneous charged lipid transfer between lipid vesicles.

    Science.gov (United States)

    Richens, Joanna L; Tyler, Arwen I I; Barriga, Hanna M G; Bramble, Jonathan P; Law, Robert V; Brooks, Nicholas J; Seddon, John M; Ces, Oscar; O'Shea, Paul

    2017-10-03

    An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

  4. Impermeability effects in three-dimensional vesicles

    International Nuclear Information System (INIS)

    Biscari, P; Canevese, S M; Napoli, G

    2004-01-01

    We analyse the effects of the impermeability constraint on the equilibrium shapes of a three-dimensional vesicle hosting a rigid inclusion. A given alteration of the inclusion and/or vesicle parameters leads to shape modifications of different orders of magnitude, when applied to permeable or impermeable vesicles. Moreover, the enclosed-volume constraint wrecks the uniqueness of stationary equilibrium shapes, and gives rise to pear-shaped or stomatocyte-like vesicles

  5. 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 ...

  6. A perspective on extracellular vesicles proteomics

    Science.gov (United States)

    Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

    2017-11-01

    Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieve from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  7. Emergence and stability of intermediate open vesicles in disk-to-vesicle transitions.

    Science.gov (United States)

    Li, Jianfeng; Zhang, Hongdong; Qiu, Feng; Shi, An-Chang

    2013-07-01

    The transition between two basic structures, a disk and an enclosed vesicle, of a finite membrane is studied by examining the minimum energy path (MEP) connecting these two states. The MEP is constructed using the string method applied to continuum elastic membrane models. The results reveal that, besides the commonly observed disk and vesicle, open vesicles (bowl-shaped vesicles or vesicles with a pore) can become stable or metastable shapes. The emergence, stability, and probability distribution of these open vesicles are analyzed. It is demonstrated that open vesicles can be stabilized by higher-order elastic energies. The estimated probability distribution of the different structures is in good agreement with available experiments.

  8. 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).

  9. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer

    OpenAIRE

    Chen, I-Hsuan; Xue, Liang; Hsu, Chuan-Chih; Paez, Juan Sebastian Paez; Pan, Li; Andaluz, Hillary; Wendt, Michael K.; Iliuk, Anton B.; Zhu, Jian-Kang; Tao, W. Andy

    2017-01-01

    Protein phosphorylation is a major regulatory mechanism for many cellular functions, but no phosphoprotein in biofluids has been developed for disease diagnosis because of the presence of active phosphatases. This study presents a general strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs from small volumes of plasma sam...

  10. Formation of Oligovesicular Vesicles by Micromanipulation

    Directory of Open Access Journals (Sweden)

    Yukihisa Okumura

    2011-09-01

    Full Text Available Cell-sized lipid bilayer membrane vesicles (giant vesicles, GVs or semi-vesicles were formed from egg yolk phosphatidylcholine on a platinum electrode under applied electric voltage by electroformation. Micromanipulation of the semi-vesicle by first pressing its membrane with a glass microneedle and then withdrawing the needle left a GV in the interior of the vesicle. During the process, an aqueous solution of Ficoll that filled the needle was introduced into the newly formed inner vesicle and remained encapsulated. Approximately 50% of attempted micromanipulation resulted in the formation of an inner daughter vesicle, “microvesiculation”. By repeating the microvesiculation process, multiple inner GVs could be formed in a single parent semi-vesicle. A semi-vesicle with inner GVs could be detached from the electrode by scraping with a microneedle, yielding an oligovesicular vesicle (OVV with desired inner aqueous contents. Microvesiculation of a GV held on the tip of a glass micropipette was also possible, and this also produced an OVV. Breaking the membrane of the parent semi-vesicle by micromanipulation with a glass needle after microvesiculation, released the inner GVs. This protocol may be used for controlled formation of GVs with desired contents.

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

    International Nuclear Information System (INIS)

    McCrea, P.D.

    1987-01-01

    Each of the five acetylcholine receptor (AChR) subunits, α 2 β-γδ, 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 δ 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 δ-δ desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of δ 2 to δ 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 3 H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space

  12. A simplified method to recover urinary vesicles for clinical applications, and sample banking.

    Science.gov (United States)

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

    2014-12-23

    Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking.

  13. 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.

  14. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    Directory of Open Access Journals (Sweden)

    Thomas Kieselbach

    Full Text Available Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs. To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e using liquid chromatography-tandem mass spectrometry (LC-MS/MS. This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

  15. Spontaneous Vesicle Recycling in the Synaptic Bouton

    Directory of Open Access Journals (Sweden)

    Sven eTruckenbrodt

    2014-12-01

    Full Text Available The trigger for synaptic vesicle exocytosis is Ca2+, which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca2+ levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca2+ sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca2+. The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs responding to Ca2+ fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

  16. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  18. Optogenetic acidification of synaptic vesicles and lysosomes.

    Science.gov (United States)

    Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2015-12-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

  19. Ultrasound-responsive ultrathin multiblock copolyamide vesicles

    Science.gov (United States)

    Huang, Lei; Yu, Chunyang; Huang, Tong; Xu, Shuting; Bai, Yongping; Zhou, Yongfeng

    2016-02-01

    This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation.This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation. Electronic supplementary information (ESI) available: Details of experiments and characterization, and FT-IR, TEM, DPD, FL and micro-DSC results. See DOI: 10.1039/c5nr08596a

  20. 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

  1. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    Felix Royo

    Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

  2. Trafficking of astrocytic vesicles in hippocampal slices

    International Nuclear Information System (INIS)

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-01-01

    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.

  3. Extracellular Vesicles in Hematological Disorders

    Directory of Open Access Journals (Sweden)

    Anat Aharon

    2014-10-01

    Full Text Available Extracellular vesicles (EVs, comprised of exosomes, microparticles, apoptotic bodies, and other microvesicles, are shed from a variety of cells upon cell activation or apoptosis. EVs promote clot formation, mediate pro-inflammatory processes, transfer proteins and miRNA to cells, and induce cell signaling that regulates cell differentiation, proliferation, migration, invasion, and apoptosis. This paper will review the contribution of EVs in hematological disorders, including hemoglobinopathies (sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, and hematological malignancies (lymphomas, myelomas, and acute and chronic leukemias.

  4. Extracellular Vesicles as Biomarkers and Therapeutics in Dermatology: A Focus on Exosomes.

    Science.gov (United States)

    McBride, Jeffrey D; Rodriguez-Menocal, Luis; Badiavas, Evangelos V

    2017-08-01

    Extracellular vesicles (exosomes, microvesicles, and apoptotic bodies) are ubiquitous in human tissues, circulation, and body fluids. Of these vesicles, exosomes are of growing interest among investigators across multiple fields, including dermatology. The characteristics of exosomes, their associated cargo (nucleic acids, proteins, and lipids), and downstream functions are vastly different, depending on the cell origin. Here, we review concepts in extracellular vesicle biology, with a focus on exosomes, highlighting recent studies in the field of dermatology. Furthermore, we highlight emerging technical issues associated with isolating and measuring exosomes. Extracellular vesicles, including exosomes, have immediate potential for serving as biomarkers and therapeutics in dermatology over the next decade. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1

    Directory of Open Access Journals (Sweden)

    Andrew I. Yao

    2011-01-01

    Full Text Available It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600. In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis.

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

    OpenAIRE

    L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, 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 co...

  7. Size Exclusion HPLC Detection of Small-Size Impurities as a Complementary Means for Quality Analysis of Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Tao Huang

    2015-07-01

    Full Text Available For extracellular vesicle research, whether for biomarker discoveries or therapeutic applications, it is critical to have high-quality samples. Both microscopy and NanoSight Tracking Analysis (NTA for size distribution have been used to detect large vesicles. However, there is currently no well-established method that is convenient for routine quality analysis of small-size impurities in vesicle samples. In this paper we report a convenient method, called ‘size-exclusion high-performance liquid chromatography’ (SE-HPLC, alongside NTA and Microscopy analysis to guide and qualify the isolation and processing of vesicles. First, the SE-HPLC analysis was used to detect impurities of small-size proteins during the ultra-centrifugation process of vesicle isolation; it was then employed to test the changes of vesicles under different pH conditions or integrity after storage. As SE-HPLC is generally accessible in most institutions, it could be used as a routine means to assist researchers in examining the integrity and quality of extracellular vesicles along with other techniques either during isolation/preparation or for further engineering and storage.

  8. 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

  9. 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.

  10. Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

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    Estelle Lebègue

    2018-02-01

    Full Text Available Polydiacetylene (PDA inserted in films or in vesicles has received increasing attention due to its property to undergo a blue-to-red colorimetric transition along with a change from non-fluorescent to fluorescent upon application of various stimuli. In this review paper, the principle for the detection of various microorganisms (bacteria, directly detected or detected through the emitted toxins or through their DNA, and viruses and of antibacterial and antiviral peptides based on these responsive PDA vesicles are detailed. The analytical performances obtained, when vesicles are in suspension or immobilized, are given and compared to those of the responsive vesicles mainly based on the vesicle encapsulation method. Many future challenges are then discussed.

  11. Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles

    International Nuclear Information System (INIS)

    Brown, R.E.; Sugar, I.P.; Thompson, T.E.

    1985-01-01

    The transfer kinetics of the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1) were investigated by monitoring tritiated asialo-GM1 movement from donor to acceptor vesicles. Two different methods were employed to separate donor and acceptor vesicles at desired time intervals. In one method, a negative charge was imparted to dipalmitoylphosphatidylcholine donor vesicles by including 10 mol% dipalmitoylphosphatidic acid. Donors were separated from neutral dipalmitoylphosphatidylcholine acceptor vesicles by ion-exchange chromatography. In the other method, small, unilamellar donor vesicles and large, unilamellar acceptor vesicles were coincubated at 45 degrees C and then separated at desired time intervals by molecular sieve chromatography. The majority of asialo-GM1 transfer to acceptor vesicles occurred as a slow first-order process with a half-time of about 24 days assuming that the relative concentration of asialo-GM1 in the phospholipid matrix was identical in each half of the donor bilayer and that no glycolipid flip-flop occurred. Asialo-GM1 net transfer was calculated relative to that of [ 14 C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. A nearly identical transfer half-time was obtained when the phospholipid matrix was changed from dipalmitoylphosphatidylcholine to palmitoyloleoylphosphatidylcholine. Varying the acceptor vesicle concentration did not significantly alter the asialo-GM1 transfer half-time. This result is consistent with a transfer mechanism involving diffusion of glycolipid through the aqueous phase rather than movement of glycolipid following formation of collisional complexes between donor and acceptor vesicles. (Abstract Truncated)

  12. Thermodynamics and kinetics of vesicles formation processes.

    Science.gov (United States)

    Guida, Vincenzo

    2010-12-15

    Vesicles are hollow aggregates, composed of bilayers of amphiphilic molecules, dispersed into and filled with a liquid solvent. These aggregates can be formed either as equilibrium or as out of equilibrium meta-stable structures and they exhibit a rich variety of different morphologies. The surprising richness of structures, the vast range of industrial applications and the presence of vesicles in a number of biological systems have attracted the interest of numerous researchers and scientists. In this article, we review both the thermodynamics and the kinetics aspects of the phenomena of formation of vesicles. We start presenting the thermodynamics of bilayer membranes formation and deformation, with the aim of deriving the conditions for the existence of equilibrium vesicles. Specifically, we use the results from continuum thermodynamics to discuss the possibility of formation of stable equilibrium vesicles, from both mixed amphiphiles and single component systems. We also link the bilayer membrane properties to the molecular structure of the starting amphiphiles. In the second part of this article, we focus on the dynamics and kinetics of vesiculation. We review the process of vesicles formation both from planar lamellar phase under shear and from isotropic micelles. In order to clarify the physical mechanisms of vesicles formation, we continuously draw a parallel between emulsification and vesiculation processes. Specifically, we compare the experimental results, the driving forces and the relative scaling laws identified for the two processes. Describing the dynamics of vesicles formation, we also discuss why non equilibrium vesicles can be formed by kinetics control and why they are meta-stable. Understanding how to control the properties, the stability and the formation process of vesicles is of fundamental importance for a vast number of industrial applications. Copyright © 2009. Published by Elsevier B.V.

  13. Royal Society Scientific Meeting: Extracellular vesicles in the tumour microenvironment.

    Science.gov (United States)

    Pink, Ryan Charles; Elmusrati, Areeg A; Lambert, Daniel; Carter, David Raul Francisco

    2018-01-05

    Cancer cells do not grow as an isolated homogeneous mass; tumours are, in fact, complex and heterogeneous collections of cancer and surrounding stromal cells, collectively termed the tumour microenvironment. The interaction between cancer cells and stromal cells in the tumour microenvironment has emerged as a key concept in the regulation of cancer progression. Understanding the intercellular dialogue in the tumour microenvironment is therefore an important goal. One aspect of this dialogue that has not been appreciated until recently is the role of extracellular vesicles (EVs). EVs are small vesicles released by cells under both normal and pathological conditions; they can transfer biological molecules between cells leading to changes in phenotype. EVs have emerged as important regulators of biological processes and can be dysregulated in diseases such as cancer; rapidly growing interest in their biology and therapeutic potential led to the Royal Society hosting a Scientific Meeting to explore the roles of EVs in the tumour microenvironment. This cross-disciplinary meeting explored examples of how aberrant crosstalk between tumour and stromal cells can promote cancer progression, and how such signalling can be targeted for diagnostic, prognostic and therapeutic benefit. In this review, and the special edition of Philosophical Transactions of the Royal Society B that follows, we will provide an overview of the content and outcomes of this exciting meeting.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'. © 2017 The Author(s).

  14. The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

    Science.gov (United States)

    Biagini, Massimiliano; Garibaldi, Manuela; Aprea, Susanna; Pezzicoli, Alfredo; Doro, Francesco; Becherelli, Marco; Taddei, Anna Rita; Tani, Chiara; Tavarini, Simona; Mora, Marirosa; Teti, Giuseppe; D'Oro, Ugo; Nuti, Sandra; Soriani, Marco; Margarit, Immaculada; Rappuoli, Rino; Grandi, Guido; Norais, Nathalie

    2015-08-01

    Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. High-speed centrifugation induces aggregation of extracellular vesicles.

    Science.gov (United States)

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

    2015-01-01

    Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

  16. High-speed centrifugation induces aggregation of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Romain Linares

    2015-12-01

    Full Text Available Plasma and other body fluids contain cell-derived extracellular vesicles (EVs, which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

  17. Vesicles Are Persistent Features of Different Plastids.

    Science.gov (United States)

    Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

    2016-10-01

    Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  19. MR imaging of the seminal vesicles

    International Nuclear Information System (INIS)

    Edson, S.B.; Hricak, H.; Chun-Fang Chang, Y.

    1987-01-01

    The seminal vesicles of 56 healthy males and 23 males with pathologic conditions were studied with a .35-T magnet and spin-echo (SE) techniques (repetition time/echo time [msec] = 500/30 and 2,000/60). The authors analyzed (1) the size and relative signal intensity of seminal vesicles compared to surrounding fat, muscle, or urine; (2) the effect of aging on the size and signal intensity of the vesicles, and (3) the appearance of the seminal vesicles in different pathologic conditions. In the transverse plane, the normal seminal vesicle measures 31 +- 7 mm in length and 17 +- 4 mm in width. Its size or signal intensity did not change significantly with age. On SE = 500/30 images the seminal vesicles were isointense with muscle; on SE = 2,000/60 images they were isointense or slightly hypointense relative to fat. MR imaging was highly sensitive for displaying seminal vesicle pathology, based on asymmetry in size and changes in signal intensities. MR imaging provides unique information but its role in pathologic conditions needs to be further explored

  20. Cargo Release from Polymeric Vesicles under Shear

    Directory of Open Access Journals (Sweden)

    Yingying Guo

    2018-03-01

    Full Text Available In this paper we study the release of cargo from polymeric nano-carriers under shear. Vesicles formed by two star block polymers— A 12 B 6 C 2 ( A B C and A 12 B 6 A 2 ( A B A —and one linear block copolymer— A 14 B 6 ( A B , are investigated using dissipative particle dynamics (DPD simulations. A - and C -blocks are solvophobic and B -block is solvophilic. The three polymers form vesicles of different structures. The vesicles are subjected to shear both in bulk and between solvophobic walls. In bulk shear, the mechanisms of cargo release are similar for all vesicles, with cargo travelling through vesicle membrane with no preferential release location. When sheared between walls, high cargo release rate is only observed with A B C vesicle after it touches the wall. For A B C vesicle, the critical condition for high cargo release rate is the formation of wall-polymersome interface after which the effect of shear rate in promoting cargo release is secondary. High release rate is achieved by the formation of solvophilic pathway allowing cargo to travel from the vesicle cavity to the vesicle exterior. The results in this paper show that well controlled target cargo release using polymersomes can be achieved with polymers of suitable design and can potentially be very useful for engineering applications. As an example, polymersomes can be used as carriers for surface active friction reducing additives which are only released at rubbing surfaces where the additives are needed most.

  1. Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment.

    Science.gov (United States)

    Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

    2017-01-01

    Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 10 10 /ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

  2. Studying extracellular vesicle transfer by a Cre-loxP method

    NARCIS (Netherlands)

    Zomer, Anoek; Steenbeek, Sander Christiaan; Maynard, Carrie; van Rheenen, Jacco

    Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological

  3. Genome Sequence of a Hyperthermophilic Archaeon, Thermococcus nautili 30-1, That Produces Viral Vesicles.

    Science.gov (United States)

    Oberto, Jacques; Gaudin, Marie; Cossu, Matteo; Gorlas, Aurore; Slesarev, Alexeï; Marguet, Evelyne; Forterre, Patrick

    2014-03-27

    Thermococcus nautili 30-1 (formerly Thermococcus nautilus), an anaerobic hyperthermophilic marine archaeon, was isolated in 1999 from a deep-sea hydrothermal vent during the Amistad campaign. Here, we present the complete sequence of T. nautili, which is able to produce membrane vesicles containing plasmid DNA. This property makes T. nautili a model organism to study horizontal gene transfer.

  4. 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.

  5. Synaptic Vesicle Endocytosis in Different Model Systems

    Directory of Open Access Journals (Sweden)

    Quan Gan

    2018-06-01

    Full Text Available Neurotransmission in complex animals depends on a choir of functionally distinct synapses releasing neurotransmitters in a highly coordinated manner. During synaptic signaling, vesicles fuse with the plasma membrane to release their contents. The rate of vesicle fusion is high and can exceed the rate at which synaptic vesicles can be re-supplied by distant sources. Thus, local compensatory endocytosis is needed to replenish the synaptic vesicle pools. Over the last four decades, various experimental methods and model systems have been used to study the cellular and molecular mechanisms underlying synaptic vesicle cycle. Clathrin-mediated endocytosis is thought to be the predominant mechanism for synaptic vesicle recycling. However, recent studies suggest significant contribution from other modes of endocytosis, including fast compensatory endocytosis, activity-dependent bulk endocytosis, ultrafast endocytosis, as well as kiss-and-run. Currently, it is not clear whether a universal model of vesicle recycling exist for all types of synapses. It is possible that each synapse type employs a particular mode of endocytosis. Alternatively, multiple modes of endocytosis operate at the same synapse, and the synapse toggles between different modes depending on its activity level. Here we compile review and research articles based on well-characterized model systems: frog neuromuscular junctions, C. elegans neuromuscular junctions, Drosophila neuromuscular junctions, lamprey reticulospinal giant axons, goldfish retinal ribbon synapses, the calyx of Held, and rodent hippocampal synapses. We will compare these systems in terms of their known modes and kinetics of synaptic vesicle endocytosis, as well as the underlying molecular machineries. We will also provide the future development of this field.

  6. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer.

    Science.gov (United States)

    Chen, I-Hsuan; Xue, Liang; Hsu, Chuan-Chih; Paez, Juan Sebastian Paez; Pan, Li; Andaluz, Hillary; Wendt, Michael K; Iliuk, Anton B; Zhu, Jian-Kang; Tao, W Andy

    2017-03-21

    The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

  7. Radiation inactivation studies of renal brush border water and urea transport

    International Nuclear Information System (INIS)

    Verkman, A.S.; Dix, J.A.; Seifter, J.L.; Skorecki, K.L.; Jung, C.Y.; Ausiello, D.A.

    1985-01-01

    Radiation inactivation was used to determine the nature and molecular weight of water and urea transport pathways in brush border membrane vesicles (BBMV) isolated from rabbit renal cortex. BBMV were frozen to -50 degrees C, irradiated with 1.5 MeV electrons, thawed, and assayed for transport or enzyme activity. The freezing process had no effect on enzyme or transport kinetics. BBMV alkaline phosphatase activity gave linear ln(activity) vs. radiation dose plots with a target size of 68 +/- 3 kDa, similar to previously reported values. Water and solute transport were measured using the stopped-flow light-scattering technique. The rates of acetamide and osmotic water transport did not depend on radiation dose (0-7 Mrad), suggesting that transport of these substances does not require a protein carrier. In contrast, urea and thiourea transport gave linear ln(activity) vs. dose curves with a target size of 125-150 kDa; 400 mM urea inhibited thiourea flux by -50% at 0 and 4.7 Mrad, showing that radiation does not affect inhibitor binding to surviving transporters. These studies suggest that BBMV urea transport requires a membrane protein, whereas osmotic water transport does not

  8. 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.

  9. 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.

  10. 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.

  11. A scenario for a genetically controlled fission of artificial vesicles

    DEFF Research Database (Denmark)

    Bönzli, Eva; Hadorn, Maik; Jørgensen, Mikkel Girke

    2011-01-01

    to vesicles (Hanczyc et al. 2003). In the present work, we developed a scenario how a genetically controlled fission of vesicles may be achieved by the synthesis of a special class of viral proteins within artificial vesicles. Because the authors already have a lot of experience in the water-in-oil emulsion...... be incorporated into vesicles, and therefore allow the synthesis of a large number of proteins (Noireaux et al. 2005). However, vesicle fission remains one of the upcoming challenges in the artificial cell project (Noireaux et al. 2011). So far, vesicle fission is implemented by applying mechanical stress...

  12. Isolation and Characterization of Chick Epiphyseal Cartilage Matrix Vesicle Proteolipid

    Science.gov (United States)

    1988-01-01

    phospholipid-phosphate complexes in diseased and normal human bone. Metab. Bone Dis. Rel. Res., 4: 151- 156. 64 Boskey AL, Timchak DM, Lane JM, Posner AS. 1980...from bovine fetal alveolar bone and dog osteosarcoma. Calcif. Tissue Int., 35: 791-797. Howell DS, Pita JC, and Alvarez J. 1976. Possible role of...at Grand Forks Air Force Base, North Dakota. In June of 1985 he entered the Post- Doctoral Periodontics program at Wilford Hall USAF Medical Center

  13. 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.

  14. Irradiation-induced fusion between giant vesicles and photoresponsive large unilamellar vesicles containing malachite green derivative.

    Science.gov (United States)

    Uda, Ryoko M; Yoshikawa, Yuki; Kitaba, Moe; Nishimoto, Noriko

    2018-07-01

    Light-initiated fusion between vesicles has attracted much attention in the research community. In particular, fusion between photoresponsive and non-photoresponsive vesicles has been of much interest in the development of systems for the delivery of therapeutic agents to cells. We have performed fusion between giant vesicles (GVs) and photoresponsive smaller vesicles containing malachite green (MG) derivative, which undergoes ionization to afford a positive charge on the molecule by irradiation. The fusion proceeds as the concentration of GV lipid increases toward equimolarity with the lipid of the smaller vesicle. It is also dependent on the molar percentage of photoionized MG in the lipid of the smaller vesicle. On the other hand, the fusion is hardly affected by the anionic component of the GV. The photoinduced fusion was characterized by two methods, involving the mixing of lipid membranes and of aqueous contents. Fluorescence microscopy revealed that irradiation triggered the fusion of a single GV with the smaller vesicles containing MG. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

    International Nuclear Information System (INIS)

    Schulz, Joseph R.; Vega-Beltran, Jose L. de la; Beltran, Carmen; Vacquier, Victor D.; Darszon, Alberto

    2004-01-01

    The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca 2+ and Na + influx and K + and H + efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba 2+ , and has a PK + /PNa + selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization

  16. Functional transferred DNA within extracellular vesicles

    International Nuclear Information System (INIS)

    Cai, Jin; Wu, Gengze; Jose, Pedro A.; Zeng, Chunyu

    2016-01-01

    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.

  17. 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.

  18. 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.

  19. Vesicle dynamics in shear and capillary flows

    International Nuclear Information System (INIS)

    Noguchi, Hiroshi; Gompper, Gerhard

    2005-01-01

    The deformation of vesicles in flow is studied by a mesoscopic simulation technique, which combines multi-particle collision dynamics for the solvent with a dynamically triangulated surface model for the membrane. Shape transitions are investigated both in simple shear flows and in cylindrical capillary flows. We focus on reduced volumes, where the discocyte shape of fluid vesicles is stable, and the prolate shape is metastable. In simple shear flow at low membrane viscosity, the shear induces a transformation from discocyte to prolate with increasing shear rate, while at high membrane viscosity, the shear induces a transformation from prolate to discocyte, or tumbling motion accompanied by oscillations between these two morphologies. In capillary flow, at small flow velocities the symmetry axis of the discocyte is found not to be oriented perpendicular to the cylinder axis. With increasing flow velocity, a transition to a prolate shape occurs for fluid vesicles, while vesicles with shear-elastic membranes (like red blood cells) transform into a coaxial parachute-like shape

  20. 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.

  1. 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.

  2. The Bretherton Problem for a Vesicle

    Science.gov (United States)

    Barakat, Joseph; Spann, Andrew; Shaqfeh, Eric

    2016-11-01

    The motion of a lipid bilayer vesicle through a circular tube is investigated by singular perturbation theory in the limit of vanishing clearance. The vesicle is treated as a sac of fluid enclosed by a thin, elastic sheet that admits a bending stiffness. It is assumed that the vesicle is axisymmetric and swollen to a near-critical volume such that the clearance "e" between the membrane and the tube wall is very small. In this limit, bending resistance is of negligible importance compared to the isotropic tension, allowing the vesicle to be treated as a "no-slip bubble." The effective membrane tension is found to scale inversely with "e" raised to the 3/2 power with a comparatively weak Marangoni gradient. The extra pressure drop is found to have a leading contribution due to the cylindrical midsection, which scales inversely with "e," as well as a correction due to the end caps, which scales inversely with the square root of "e." The apparent viscosity is predicted as a unique function of the geometry. The theory exhibits excellent agreement with a simplified, "quasi-parallel" theory and with direct numerical simulations using the boundary element method. The results of this work are compared to those for bubbles, rigid particles, and red blood cells in confined flows.

  3. Nanoplasmonic ruler to measure lipid vesicle deformation

    Czech Academy of Sciences Publication Activity Database

    Jackman, J.A.; Špačková, Barbora; Linardy, E.; Kim, M.C.; Yoon, B.K.; Homola, Jiří; Cho, N.J.

    2016-01-01

    Roč. 52, č. 1 (2016), s. 76-79 ISSN 1359-7345 R&D Projects: GA ČR(CZ) GBP205/12/G118 Institutional support: RVO:67985882 Keywords : nanomaterial * silicon * lipid vesicle Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 6.319, year: 2016

  4. Electrogenic sulfate uptake by crustacean hepatopancreatic basolateral membrane vesicles

    International Nuclear Information System (INIS)

    Cattey, M.A.; Gerencser, G.A.; Aheam, G.A.

    1990-01-01

    Basolateral membrane vesicles (BLMV) were isolated from Atlantic lobster (Homarus americanus) hepatopancreas and purified by discontinuous sucrose gradient centrifugation. BLMV prepared in this fashion were osmotically reactive exhibiting linear dependence of vesicular 35 SO 4 -2 uptake to increasing external osmotic pressure with negligible non-specific isotope binding. Under short circuited conditions (valinomycin/K + ) BLMV responded to either a HCO 3 - gradient directed out or equilibrated HCO 3 - (10 mM) by displaying short term accumulation of sulfate above that of equilibrium. Uptake of divalent anion was unaffected by an inwardly directed transmembrane Na + or tetramethylammonium + gradient. 35 SO 4 -2 /HCO 3 - exchange in the presence of valinomycin was stimulated by transient inside positive K + diffusion potentials and inhibited by transient inside negative K + diffusion potentials. The role of electrogenic anion exchange by hepatopancreas BLMV in transcellular sulfate transport is discussed

  5. 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.

  6. Calcium transport in sealed vesicles from red beet (Beta vulgaris L.) storage tissue. II. Characterization of 45Ca2+ uptake into plasma membrane vesicles

    International Nuclear Information System (INIS)

    Giannini, J.L.; Ruiz-Cristin, J.; Briskin, D.P.

    1987-01-01

    Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using 45 Ca 2+ . Uptake of 45 Ca 2+ by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of 45 Ca 2+ uptake to ATP utilization via ΔμH + , no evidence for a secondary H + /Ca 2+ antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca 2+ and an imposed pH gradient could not drive 45 Ca 2+ uptake. Optimal uptake of 45 Ca 2+ occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of 45 Ca 2+ uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K/sub m/ values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving 45 Ca 2+ uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell

  7. Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

    Science.gov (United States)

    Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

    2017-01-01

    Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright

  8. 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....

  9. Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

    Directory of Open Access Journals (Sweden)

    Bernard Thay

    Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

  10. Effects of Silodosin and Tamsulosin on the Seminal Vesicle Contractile Response.

    Science.gov (United States)

    Hayashi, Tokumasa; Takeya, Mitsue; Nakamura, Kei-ichiro; Matsuoka, Kei

    2016-01-01

    To understand the mechanisms underlying ejaculation dysfunction caused by α1A-adrenocetor (AR) antagonists, the effects of α1A-AR antagonists on the contractile responses of the seminal vesicle were investigated. Isolated seminal vesicles from guinea pigs were cannulated and pressurized, and the changes in the intraluminal pressure were recorded. Periodic applications of electrical stimulation (ES) caused biphasic increase in the intraluminal pressure, that is, initial and subsequent contractions. The effects of silodosin and tamsulosin, α1A-AR antagonists, on the contractile responses were examined. The ES-induced biphasic contractions were blocked by tetrodotoxin (TTX). Silodosin and tamsulosin suppressed the initial contractions in a dose-dependent manner, while also exerting various inhibitory effects on the subsequent contractions. Increases in the intraluminal pressure facilitated spontaneous phasic contractions. The spontaneous contractions were not affected by TTX or α1A-AR antagonists, but were abolished by nifedipine. The initial contractions triggered by neuronal excitations were suppressed by silodosin and tamsulosin, suggesting that the ejaculation dysfunction may be attributed to the α1A-AR antagonist-mediated suppression of nerve-evoked contractions in the seminal vesicle. The subsequent contractions may be induced by mechanical stimulation associated with the initial, nerve-evoked contractions. Alternatively, other transmitters may be involved to various degrees in the neuromuscular transmission of the seminal vesicle. © 2014 Wiley Publishing Asia Pty Ltd.

  11. SNX9 - a prelude to vesicle release.

    Science.gov (United States)

    Lundmark, Richard; Carlsson, Sven R

    2009-01-01

    The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

  12. Stem cell extracellular vesicles and kidney injury

    OpenAIRE

    Grange, Cristina; Iampietro, Corinne; Bussolati, Benedetta

    2017-01-01

    Extracellular vesicles (EVs) appear as a new promising cell-free therapy for acute and chronic renal diseases. EVs retain characteristics of the cell of origin and those derived from stem cells may mimic their regenerative properties per se. In fact, EVs contain many active molecules such as proteins and RNA species that act on target cells through different mechanisms, stimulating proliferation and angiogenesis and reducing apoptosis and inflammation. There are several reports that demonstra...

  13. 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

  14. High energy irradiation of bacterial membrane vesicles

    International Nuclear Information System (INIS)

    De La Rosa, M.A.M.

    1977-01-01

    The interactions of membrane components and two well-defined transport systems in the E. coli ML 308-225 membrane vesicles with 60 Co gamma radiation were investigated. The results presented show that gamma radiation can monitor membrane components and functions of varying radiosensitivities. The possible application of high-energy radiation as a physical probe of membrane structure and functions is indeed promising

  15. Viscoelastic deformation of lipid bilayer vesicles.

    Science.gov (United States)

    Wu, Shao-Hua; Sankhagowit, Shalene; Biswas, Roshni; Wu, Shuyang; Povinelli, Michelle L; Malmstadt, Noah

    2015-10-07

    Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic.

  16. 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.

  17. Release of canine parvovirus from endocytic vesicles

    International Nuclear Information System (INIS)

    Suikkanen, Sanna; Antila, Mia; Jaatinen, Anne; Vihinen-Ranta, Maija; Vuento, Matti

    2003-01-01

    Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A 2 like domain in N-terminus of VP1. In this study we characterized the role of PLA 2 activity on CPV entry process. PLA 2 activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA 2 inhibitors inhibited the viral proliferation suggesting that PLA 2 activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA 2 activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A 1 , brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A 1 , brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA 2 activity of the virus. These results suggest that parvoviral PLA 2 activity is essential for productive infection and

  18. Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

    Science.gov (United States)

    Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

    2014-10-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    International Nuclear Information System (INIS)

    Abu-Hamdah, Rania; Cho, Won Jin; Hoerber, J.K.H.; Jena, Bhanu P.

    2006-01-01

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 μm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles

  20. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Hamdah, Rania [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Cho, Won Jin [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Hoerber, J.K.H. [Department of Physics, University of Bristol, Bristol BS8 1TD (United Kingdom); Jena, Bhanu P. [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States)]. E-mail: bjena@med.wayne.edu

    2006-06-15

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 {mu}m in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

  1. Bending Elasticity Modulus of Giant Vesicles Composed of Aeropyrum Pernix K1 Archaeal Lipid

    Directory of Open Access Journals (Sweden)

    Julia Genova

    2015-03-01

    Full Text Available Thermally induced shape fluctuations were used to study elastic properties of giant vesicles composed of archaeal lipids C25,25-archetidyl (glucosyl inositol and C25,25-archetidylinositol isolated from lyophilised Aeropyrum pernix K1 cells. Giant vesicles were created by electroformation in pure water environment. Stroboscopic illumination using a xenon flash lamp was implemented to remove the blur effect due to the finite integration time of the camera and to obtain an instant picture of the fluctuating vesicle shape. The mean weighted value of the bending elasticity modulus kc of the archaeal membrane determined from the measurements meeting the entire set of qualification criteria was (1.89 ± 0.18 × 10−19 J, which is similar to the values obtained for a membrane composed of the eukaryotic phospholipids SOPC (1.88 ± 0.17 × 10−19 J and POPC (2.00 ± 0.21 ´ 10−19 J. We conclude that membranes composed of archaeal lipids isolated from Aeropyrum pernix K1 cells have similar elastic properties as membranes composed of eukaryotic lipids. This fact, together with the importance of the elastic properties for the normal circulation through blood system, provides further evidence in favor of expectations that archaeal lipids could be appropriate for the design of drug delivery systems.

  2. Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

    Directory of Open Access Journals (Sweden)

    Débora L Oliveira

    2010-06-01

    Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

  3. 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.

  4. Deformation of phospholipid vesicles in an optical stretcher

    OpenAIRE

    Delabre , Ulysse; Feld , Kasper; Crespo , Eleonore; Whyte , Graeme; Sykes , Cecile; Seifert , Udo; Guck , Jochen

    2015-01-01

    International audience; Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelength...

  5. 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.

  6. 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...

  7. Uptake of 75Se-selenite by brush border membrane vesicles from chick duodenum stimulated by vitamin D

    International Nuclear Information System (INIS)

    Mykkanen, H.M.; Wasserman, R.H.

    1989-01-01

    Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of 75Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of 75Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60-120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional rate of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4-100 microM Se. 75Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 Iu, 72 h) or with 1,25(OH)2-cholecalciferol (0.5 microgram given 16 h prior to isolation of the vesicles) significantly enhanced 75Se uptake. A threefold excess of mannitol in the outside buffer reduced 75Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with 75Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with 75Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane

  8. Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3

    Directory of Open Access Journals (Sweden)

    Johannes eZimmermann

    2015-09-01

    Full Text Available The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3 is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron’s characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles. We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

  9. ABC Triblock Copolymer Vesicles with Mesh-like Morphology

    Science.gov (United States)

    Zhao, Wei; Russell, Thomas; Grason, Gregory

    2010-03-01

    Polymer vesicles can be made from poly(isoprene-b-styrene-b-2-vinylpyridene) (PI-b-PS-b-P2VP) triblock copolymer under the confinement of anodic aluminum oxide (AAO) membrane. It was found that these vesicles have well-defined, nanoscopic size and a microphase-separated hydrophobic core, comprised of PS and PI blocks. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the core at a well-defined composition of three blocks. Confinement played an important role in generating these vesicles with such an unusual morphology.

  10. Sugar-Decorated Sugar Vesicles : Lectin-Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

    NARCIS (Netherlands)

    Voskuhl, Jens; Stuart, Marc C. A.; Ravoo, Bart Jan

    2010-01-01

    An artificial glycocalix self-assembles when unilamellar bilayer vesicles of amphiphilic beta-cyclodextrins are decorated with maltose and lactose by host-guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both

  11. Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate

    DEFF Research Database (Denmark)

    Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias

    2015-01-01

    supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced......-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release....

  12. Nonenzymatic glycation of phosphatidylethanolamine in erythrocyte vesicles

    International Nuclear Information System (INIS)

    Patkowska, M.J.; Horowitz, M.I.

    1986-01-01

    Unsealed inside-out and right-side out vesicles were prepared from human red cells. The vesicles were incubated with D-glucose [ 14 C(U)] and sodium cyanoborohydride in phosphate buffer, pH 7.4. After incubation, lipids were extracted with 1-butanol and non-lipid contaminants removed by Sephadex G-25 chromatography. Phosphatidylethanolamine-sorbitol was purified by chromatography on columns of silicic acid and phenylboronate agarose gel. Phospholipase C (B. cereus) liberated phosphoethanolamine-sorbitol (I) which comigrated on TLC with synthetic I prepared by reductive condensation of phosphoethanolamine and D-glucose and also with the product of phospholipase C (B. cereus) hydrolysis of reference phosphatidylethanolamine-sorbitol. Exposure of I to alkaline phosphatase (E. coli) gave P/sub i/ and ethanolamine-sorbitol (II) which comigrated on TLC with synthetic II prepared by reductive condensation of ethanolamine and D-glucose or by phospholipase D hydrolysis of reference phosphatidylethanolamine-sorbitol. These studies demonstrate that vesicular phosphatidylethanolamine can be reductively glycated and illustrate the applicability of both phospholipase C and phospholipase D in characterizing glycated phosphoglycerides

  13. Mechanical collapse of confined fluid membrane vesicles.

    Science.gov (United States)

    Rim, Jee E; Purohit, Prashant K; Klug, William S

    2014-11-01

    Compact cylindrical and spherical invaginations are common structural motifs found in cellular and developmental biology. To understand the basic physical mechanisms that produce and maintain such structures, we present here a simple model of vesicles in confinement, in which mechanical equilibrium configurations are computed by energy minimization, balancing the effects of curvature elasticity, contact of the membrane with itself and the confining geometry, and adhesion. For cylindrical confinement, the shape equations are solved both analytically and numerically by finite element analysis. For spherical confinement, axisymmetric configurations are obtained numerically. We find that the geometry of invaginations is controlled by a dimensionless ratio of the adhesion strength to the bending energy of an equal area spherical vesicle. Larger adhesion produces more concentrated curvatures, which are mainly localized to the "neck" region where the invagination breaks away from its confining container. Under spherical confinement, axisymmetric invaginations are approximately spherical. For extreme confinement, multiple invaginations may form, bifurcating along multiple equilibrium branches. The results of the model are useful for understanding the physical mechanisms controlling the structure of lipid membranes of cells and their organelles, and developing tissue membranes.

  14. Getting there: vesicles en route for plant cytokinesis

    NARCIS (Netherlands)

    Ozdoba, A.

    2007-01-01

    In dividing plant cells, membranous vesicles (60-80 nm in diameter) are transported to the site where a new cell wall that separates the daughter cells is formed. In this thesis the physical parameters size and stiffness that vesicles require to reach the forming cell plate were studied. Synthetic

  15. Spontaneous transfer of ganglioside GM1 between phospholipid vesicles

    International Nuclear Information System (INIS)

    Brown, R.E.; Thompson, T.E.

    1987-01-01

    The transfer kinetics of the negatively charged glycosphingolipid II 3 -N-acetylneuraminosyl-gangliotetraosylceramide (GM 1 ) were investigated by monitoring tritiated GM 1 movement between donor and acceptor vesicles. After appropriate incubation times at 45 0 C, donor and acceptor vesicles were separated by molecular sieve chromatography. Donors were small unilamellar vesicles produced by sonication, whereas acceptors were large unilamellar vesicles produced by either fusion or ethanol injection. Initial GM 1 transfer to acceptors followed first-order kinetics with a half-time of about 40 h assuming that GM 1 is present in equal mole fractions in the exterior and interior surfaces of the donor vesicle bilayer and that no glycolipid flip-flop occurs. GM 1 net transfer was calculated relative to that of [ 14 C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. Factors affecting the GM 1 interbilayer transfer rate included phospholipid matrix composition, initial GM 1 concentration in donor vesicles, and the GM 1 distribution in donor vesicles with respect to total lipid symmetry. The findings provide evidence that GM 1 is molecularly dispersed at low concentrations within liquid-crystalline phospholipid bilayers

  16. Loading of Vesicles into Soft Amphiphilic Nanotubes using Osmosis

    NARCIS (Netherlands)

    Erne, Petra M.; van Bezouwen, Laura S.; Stacko, Peter; van Dtjken, Derk Jan; Chen, Jiawen; Stuart, Marc C. A.; Boekema, Eghert J.; Feringa, Ben L.

    2015-01-01

    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

  17. Slow sedimentation and deformability of charged lipid vesicles.

    Directory of Open Access Journals (Sweden)

    Iván Rey Suárez

    Full Text Available 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.

  18. Model of separated form factors for unilamellar vesicles

    International Nuclear Information System (INIS)

    Kiselev, M.A.; Aksenov, V.L.; Lesieur, P.; Lombardo, D.; Kiselev, A.M.

    2001-01-01

    A new model of separated form factors is proposed for the evaluation of small-angle neutron scattering curves from large unilamellar vesicles. The validity of the model was checked via comparison with the model of a hollow sphere. The model of separated form factors and the hollow sphere model give a reasonable agreement in the evaluation of vesicle parameters

  19. Molecular dynamics simulations of lipid vesicle fusion in atomic detail

    NARCIS (Netherlands)

    Knecht, Volker; Marrink, Siewert-Jan

    The fusion of a membrane-bounded vesicle with a target membrane is a key step in intracellular trafficking, exocytosis, and drug delivery. Molecular dynamics simulations have been used to study the fusion of small unilamellar vesicles composed of a dipalmitoyl-phosphatidylcholine (DPPC)/palmitic

  20. The freezing process of small lipid vesicles at molecular resolution

    NARCIS (Netherlands)

    Risselada, H. Jelger; Marrink, Siewert J.

    2009-01-01

    At present very little is known about the kinetic barriers which a small vesicle will face during the transformation from the liquid-crystalline to the gel phase, and what the structure of frozen vesicles looks like at the molecular level. The formation of gel domains in the strongly curved bilayer

  1. Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

    NARCIS (Netherlands)

    Jackson, R.L.; Verkleij, A.J.; Zoelen, E.J.J. van; Lane, L.K.; Schwartz, A.; Deenen, L.L.M. van

    Purified lamb kidney Na+, K+-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the Mτ- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles

  2. Membrane Protrusion Coarsening and Nanotubulation within Giant Unilamellar Vesicles

    KAUST Repository

    Węgrzyn, Ilona

    2011-11-16

    Hydrophobic side groups on a stimuli-responsive polymer, encapsulated within a single giant unilamellar vesicle, enable membrane attachment during compartment formation at elevated temperatures. We thermally modulated the vesicle through implementation of an IR laser via an optical fiber, enabling localized directed heating. Polymer-membrane interactions were monitored using confocal imaging techniques as subsequent membrane protrusions occurred and lipid nanotubes formed in response to the polymer hydrogel contraction. These nanotubes, bridging the vesicle membrane to the contracting hydrogel, were retained on the surface of the polymer compartment, where they were transformed into smaller vesicles in a process reminiscent of cellular endocytosis. This development of a synthetic vesicle system containing a stimuli-responsive polymer could lead to a new platform for studying inter/intramembrane transport through lipid nanotubes. © 2011 American Chemical Society.

  3. 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.

  4. Ganglioside GM1 spontaneous transfer between phospholipid vesicles

    International Nuclear Information System (INIS)

    Brown, R.E.; Sugar, I.P.; Thompson, T.E.

    1986-01-01

    The transfer kinetics of the monosiaylated glycosphingolipid, GM 1 , between different size phospholipid vesicles was measured using molecular sieve chromatography. At desired time intervals, small unilamellar donor vesicles were separated from large unilamellar acceptor vesicles by elution from a Sephacryl S-500 column [ 3 H]-GM 1 net transfer was calculated relative to [ 14 C]-cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. The initial GM 1 transfer rate between 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles at 45 0 C deviated slightly from first order kinetics and possessed a half time of 3.6 days. This transfer half time is an order of magnitude shorter than that observed from the desiaylated derivative of GM 1 . The transfer kinetics are consistent with the authors recent electron microscopic results suggesting a molecular distribution of GM 1 in liquid-crystalline phosphatidylcholine bilayers

  5. Interaction of a potyviral VPg with anionic phospholipid vesicles

    International Nuclear Information System (INIS)

    Rantalainen, Kimmo I.; Christensen, Peter A.; Hafren, Anders; Otzen, Daniel E.; Kalkkinen, Nisse; Maekinen, Kristiina

    2009-01-01

    The viral genome-linked protein (VPg) of Potato virus A (PVA) is a multifunctional protein that belongs to a class of intrinsically disordered proteins. Typically, this type of protein gains a more stable structure upon interactions or posttranslational modifications. In a membrane lipid strip overlay binding assay, PVA VPg was found to bind phosphatidylserine (PS), but not phosphatidylcholine (PC). According to circular dichroism spectroscopy, the secondary structure of PVA VPg was stabilized upon interactions with PS and phosphatidylglycerol (PG), but not with PC vesicles. It is possible that this stabilization favored the formation of α-helical structures. Limited tryptic digestion showed that the interaction with anionic vesicles protected certain, otherwise accessible, trypsin cleavage sites. An electron microscopy study revealed that interaction with VPg substantially increased the vesicle diameter and caused the formation of pore or plaque-like electron dense spots on the vesicle surface, which gradually led to disruption of the vesicles.

  6. The role of extracellular vesicles in parasite-host interaction

    Directory of Open Access Journals (Sweden)

    Justyna Gatkowska

    2016-09-01

    Full Text Available Extracellular vesicles (EVs, initially considered cell debris, were soon proved to be an essential tool of intercellular communication enabling the exchange of information without direct contact of the cells. At present EVs are the subject of extensive research due to their universal presence in single- and multi-cell organisms, regardless of their systematic position, and their substantial role in cell-to-cell communication. EVs seem to be released by both prokaryotic and eukaryotic cells under natural (in vivo and laboratory (in vitro conditions. Even purified fractions of isolated EVs comprise various membrane-derived structures. However, EVs can be classified into general groups based primarily on their size and origin. EVs may carry various materials, and ongoing research investigations give new insight into their potenti participation in critical biological processes, e.g. carcinogenesis. This paper presents current knowledge on the EVs’ involvement in host–parasite interactions including the invasion process, the maintenance of the parasite infection and modulation of the host immune response to parasite antigenic stimulation, as well as perspectives of the potential use of EVs as immunoprophylactic and diagnostic tools for controlling parasite infections. The most numerous literature data concern protozoan parasites, especially those of the greatest medical and social importance worldwide. However, available information about the EVs’ contribution to helminth invasion has also been included.

  7. Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.

    Science.gov (United States)

    Chitcholtan, Kenny; Hampton, Mark B; Keenan, Jacqueline I

    2008-12-01

    Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

  8. 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.

  9. Effect of diet on insulin binding and glucose transport in rat sarcolemmal vesicles

    International Nuclear Information System (INIS)

    Grimditch, G.K.; Barnard, R.J.; Sternlicht, E.; Whitson, R.H.; Kaplan, S.A.

    1987-01-01

    The purpose of this study was to compare the effects of a high-fat, high-sucrose diet (HFS) and a low-fat, high-complex carbohydrate diet (LFC) on glucose tolerance, insulin binding, and glucose transport in rat skeletal muscle. During the intravenous glucose tolerance test, peak glucose values at 5 min were significantly higher in the HFS group; 0-, 20-, and 60-min values were similar. Insulin values were significantly higher in the HFS group at all time points (except 60 min), indicating whole-body insulin resistance. Skeletal muscle was responsible, in part, for this insulin resistance, because specific D-glucose transport in isolated sarcolemmal (SL) vesicles under basal conditions was similar between LFC and HFS rats, despite the higher plasma insulin levels. Scatchard analyses of insulin binding curves to sarcolemmal vesicles revealed that the K/sub a/ of the high-affinity binding sites was significantly reduced by the HFS diet; no other binding changes were noted. Specific D-glucose transport in SL vesicles after maximum insulin stimulation (1 U/kg) was significantly depressed in the HFS group, indicating that HFS feeding also caused a postbinding defect. These results indicate that the insulin resistance in skeletal muscle associated with a HFS diet is due to both a decrease in the K/sub a/ of the high-affinity insulin receptors and a postbinding defect

  10. Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles.

    Science.gov (United States)

    Moche, Martin; Stremlau, Stefanie; Hecht, Lars; Göbel, Cornelia; Feussner, Ivo; Stöhr, Christine

    2010-01-01

    Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10-15 mM nitrate.

  11. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting

    Directory of Open Access Journals (Sweden)

    Oscar P. B. Wiklander

    2015-04-01

    Full Text Available Extracellular vesicles (EVs have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

  12. Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials

    Science.gov (United States)

    Oglęcka, Kamila; Rangamani, Padmini; Liedberg, Bo; Kraut, Rachel S; Parikh, Atul N

    2014-01-01

    Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressure and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment. DOI: http://dx.doi.org/10.7554/eLife.03695.001 PMID:25318069

  13. Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility.

    Science.gov (United States)

    Nojimoto, Fernanda D; Piffer, Renata C; Kiguti, Luiz Ricardo de A; Lameu, Claudiana; de Camargo, Antônio C M; Pereira, Oduvaldo C M; Pupo, André S

    2009-09-15

    Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.

  14. Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility

    International Nuclear Information System (INIS)

    Nojimoto, Fernanda D.; Piffer, Renata C.; Kiguti, Luiz Ricardo de A.; Lameu, Claudiana; Camargo, Antonio C.M. de; Pereira, Oduvaldo C.M.; Pupo, Andre S.

    2009-01-01

    Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg -1 , ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca 2+ transients. Sibutramine 5 and 20 mg kg -1 reduced ejaculation latency whereas 50 mg kg -1 increased ejaculation latency. Sibutramine 3 to 30 μM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 μM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl 2 . In HEK293 cells, sibutramine 10 to 100 μM inhibited intracellular Ca 2+ transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca 2+ may be involved in the delayed ejaculation observed with high doses of sibutramine.

  15. Gas Vesicle Nanoparticles for Antigen Display

    Directory of Open Access Journals (Sweden)

    Shiladitya DasSarma

    2015-09-01

    Full Text Available Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs. GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications.

  16. Morphometric image analysis of giant vesicles

    DEFF Research Database (Denmark)

    Husen, Peter Rasmussen; Arriaga, Laura; Monroy, Francisco

    2012-01-01

    We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicles...... (GUVs), a dedicated 3D-image analysis, and a quantitative analysis based in equilibrium thermodynamic considerations. This approach was tested in GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-palmitoyl-sn-glycero-3-phosphocholine/cholesterol. In general, our results show a reasonable...... agreement with previously reported data obtained by other methods. For example, our computed tie lines were found to be nonhorizontal, indicating a difference in cholesterol content in the coexisting phases. This new, to our knowledge, analytical strategy offers a way to further exploit fluorescence...

  17. Periodic-cylinder vesicle with minimal energy

    International Nuclear Information System (INIS)

    Xiao-Hua, Zhou

    2010-01-01

    We give some details about the periodic cylindrical solution found by Zhang and Ou-Yang in [1996 Phys. Rev. E 53 4206] for the general shape equation of vesicle. Three different kinds of periodic cylindrical surfaces and a special closed cylindrical surface are obtained. Using the elliptic functions contained in mathematic, we find that this periodic shape has the minimal total energy for one period when the period–amplitude ratio β ≈ 1.477, and point out that it is a discontinuous deformation between plane and this periodic shape. Our results also are suitable for DNA and multi-walled carbon nanotubes (MWNTs). (cross-disciplinary physics and related areas of science and technology)

  18. Seminal vesicle intrafraction motion analysed with cinematic magnetic resonance imaging

    International Nuclear Information System (INIS)

    Gill, Suki; Dang, Kim; Fox, Chris; Bressel, Mathias; Kron, Tomas; Bergen, Noelene; Ferris, Nick; Owen, Rebecca; Chander, Sarat; Tai, Keen Hun; Foroudi, Farshad

    2014-01-01

    This study analyses seminal vesicle displacement relative to the prostate and in relation to treatment time. A group of eleven patients undergoing prostate cancer radiotherapy were imaged with a continuous 3 T cine-MRI in the standard treatment setup position. Four images were recorded every 4 seconds for 15 minutes in the sagittal plane and every 6.5 seconds for 12 minutes in the coronal plane. The prostate gland and seminal vesicles were contoured on each MRI image. The coordinates of the centroid of the prostate and seminal vesicles on each image was analysed for displacement against time. Displacements between the 2.5 percentile and 97.5 percentile (i.e. the 2.5% trimmed range) for prostate and seminal vesicle centroid displacements were measured for 3, 5, 10 and 15 minutes time intervals in the anterior-posterior (AP), left-right (LR) and superior-inferior (SI) directions. Real time prostate and seminal vesicle displacement was compared for individual patients. The 2.5% trimmed range for 3, 5, 10 and 15 minutes for the seminal vesicle centroids in the SI direction measured 4.7 mm; 5.8 mm; 6.5 mm and 7.2 mm respectively. In the AP direction, it was 4.0 mm, 4.5 mm, 6.5 mm, and 7.0 mm. In the LR direction for 3, 5 and 10 minutes; for the left seminal vesicle, it was 2.7 mm, 2.8 mm, 3.4 mm and for the right seminal vesicle, it was 3.4 mm, 3.3 mm, and 3.4 mm. The correlation between the real-time prostate and seminal vesicle displacement varied substantially between patients indicating that the relationship between prostate displacement and seminal vesicles displacement is patient specific with the majority of the patients not having a strong relationship. Our study shows that seminal vesicle motion increases with treatment time, and that the prostate and seminal vesicle centroids do not move in unison in real time, and that an additional margin is required for independent seminal vesicle motion if treatment localisation is to the prostate

  19. A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.

    Science.gov (United States)

    Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C D; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

    2016-01-01

    The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

  20. Accumulation of low density lipoprotein associated cholesterol in calcifying vesicle fractions correlates with intimal thickening in thoracic aortas of juvenile rabbits fed a supplemental cholesterol diet

    Directory of Open Access Journals (Sweden)

    Culley Nathan C

    2006-10-01

    Full Text Available Abstract Background It has been shown that calcifying vesicles play an important role in aortic calcification and that cholesterol content in the isolated vesicle fraction is increased when rabbits are fed supplemental cholesterol diets. Whether lipoprotein-associated cholesterols and other lipids are also increased in the vesicle fraction and whether the increase correlates with atherosclerosis remain unknown. Results Fourteen juvenile male rabbits fed an atherogenic diet containing 0.5% cholesterol and 2% peanut oil for 3 months developed varying degrees of hypercholesterolemia and intimal thickening in the ascending thoracic aorta. The correlation between these two parameters was insignificant, and likely attributable to the use of small numbers of rabbits in this study. Despite this lack of correlation, we demonstrate that the accumulation of cholesterol in calcifying vesicle fractions obtained from the collagenase-digested aorta fragments correlates well with intimal thickening (r2 = 0.98, p Conclusion When limited numbers of rabbits are used, LDL-C accumulation in calcifying vesicle fractions is a better biomarker for atherosclerosis than LDL-C levels in the serum. The close association of LDL-C with calcifying vesicles may play an important role in atherosclerosis and calcification.

  1. Characterization of Extracellular Vesicles by Size-Exclusion High-Performance Liquid Chromatography (HPLC).

    Science.gov (United States)

    Huang, Tao; He, Jiang

    2017-01-01

    Extracellular vesicles (EVs) have recently attracted substantial attention due to the potential diagnostic and therapeutic relevance. Although a variety of techniques have been used to isolate and analyze EVs, it is still far away from satisfaction. Size-exclusion chromatography (SEC), which separates subjects by size, has been widely applied in protein purification and analysis. The purpose of this chapter is to show the applications of size-exclusion high-performance liquid chromatography (HPLC) as methods for EV characterization of impurities or contaminants of small size, and thus for quality assay for the purity of the samples of EVs.

  2. Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

    Directory of Open Access Journals (Sweden)

    Azusa Oshima

    2017-09-01

    Full Text Available The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs to control the fusion process. We combined large unilamellar vesicles (LUVs containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO2 substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.

  3. Formation of Giant Protein Vesicles by a Lipid Cosolvent Method

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Vararattanavech, Ardcharaporn; Vissing, Thomas

    2011-01-01

    This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent‐driven fusion of large vesicles (0.1–0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein‐reconstituted large unilamellar vesicles (LUVs)...... of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform....

  4. 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

  5. Improved Methods of Producing and Administering Extracellular Vesicles | Poster

    Science.gov (United States)

    An efficient method of producing purified extracellular vesicles (EVs), in conjunction with a method that blocks liver macrophages from clearing EVs from the body, has produced promising results for the use of EVs in cancer therapy.

  6. EVpedia : a community web portal for extracellular vesicles research

    NARCIS (Netherlands)

    Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W M; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E; Buée, Luc; Buzás, Edit I; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S; Desiderio, Dominic M; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M; Gardiner, Chris; Giebel, Bernd; Greening, David W; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F; Hill, Michelle M; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; Del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I; Rodrigues, Marcio L; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond|info:eu-repo/dai/nl/212909509; Sharma, Shivani; Siljander, Pia; Simpson, Richard J; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song; Nolte - t Hoen, Esther|info:eu-repo/dai/nl/261632175

    2014-01-01

    MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We

  7. EVpedia: a community web portal for extracellular vesicles research

    NARCIS (Netherlands)

    Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W. M.; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E.; Buée, Luc; Buzás, Edit I.; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S.; Desiderio, Dominic M.; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M.; Gardiner, Chris; Giebel, Bernd; Greening, David W.; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F.; Hill, Michelle M.; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V.; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N.; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I.; Rodrigues, Marcio L.; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J.; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J.; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

    2015-01-01

    Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. We present an improved

  8. Biological properties of extracellular vesicles and their physiological functions

    NARCIS (Netherlands)

    Yáñez-Mó, María; Siljander, Pia R-M; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E; Buzas, Edit I; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Cordeiro-da Silva, Anabela; Fais, Stefano; Falcon-Perez, Juan M; Ghobrial, Irene M; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H H; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Nolte-'t Hoen, Esther N M; Nyman, Tuula A; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; Del Portillo, Hernando A; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem|info:eu-repo/dai/nl/074352385; Stukelj, Roman; Van der Grein, Susanne G|info:eu-repo/dai/nl/412755211; Vasconcelos, M Helena; Wauben, Marca H M|info:eu-repo/dai/nl/112675735; De Wever, Olivier

    2015-01-01

    In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological

  9. Packing states of multilamellar vesicles in a nonionic surfactant system

    DEFF Research Database (Denmark)

    Le, T.D.; Olsson, U.; Mortensen, K.

    2001-01-01

    -alpha(*) phase using the noninvasive small-angle neutron scattering (SANS) technique, one while heating and the other while cooling the sample. Data from the heating and cooling cycles were used to demonstrate reversibility of the system. Three states of packing can be identified from the scattering profiles......Lyotropic lamellar phases under shear flow have been shown to form multilamellar vesicles (MLVs), an onion-like structure. The size of the vesicles is governed by the shear imposed on the sample. Previously, we studied the structural transformation from multilamellar vesicles to lamellae to sponge...... under shear. Here, we focused only in the MLV region, L-alpha(*), of a temperature sensitive surfactant system (C12E4-water) to investigate the packing of multilamellar vesicles as a function of temperature under constant shear. Two sets of temperature scan experiments were performed in the L...

  10. Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

    Science.gov (United States)

    Xie, H

    2015-01-01

    Porphyromonas gingivalis is one of the keystone pathogens associated with chronic periodontitis. All P. gingivalis strains examined thus far produce outer membrane vesicles. Recent studies have found that vesicles possess some well-known virulence factors of P. gingivalis such as adhesins, toxins and proteolytic enzymes. Carrying most of the characteristic features of their parent P. gingivalis cells, vesicles communicate with host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of pathogenic bacteria-dominated microbial communities. An in-depth understanding of both the nature and role of vesicles in the pathogenicity of P. gingivalis is both important and timely, particularly when speaking of periodontitis and its related systemic effects. PMID:26343879

  11. Integral equation methods for vesicle electrohydrodynamics in three dimensions

    Science.gov (United States)

    Veerapaneni, Shravan

    2016-12-01

    In this paper, we develop a new boundary integral equation formulation that describes the coupled electro- and hydro-dynamics of a vesicle suspended in a viscous fluid and subjected to external flow and electric fields. The dynamics of the vesicle are characterized by a competition between the elastic, electric and viscous forces on its membrane. The classical Taylor-Melcher leaky-dielectric model is employed for the electric response of the vesicle and the Helfrich energy model combined with local inextensibility is employed for its elastic response. The coupled governing equations for the vesicle position and its transmembrane electric potential are solved using a numerical method that is spectrally accurate in space and first-order in time. The method uses a semi-implicit time-stepping scheme to overcome the numerical stiffness associated with the governing equations.

  12. Extracellular vesicles provide a means for tissue crosstalk during exercise

    DEFF Research Database (Denmark)

    Whitham, Martin; Parker, Benjamin L; Friedrichsen, Martin

    2018-01-01

    Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative prot...

  13. EVpedia : A community web portal for extracellular vesicles research

    NARCIS (Netherlands)

    Kim, Dae Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si Hyun; Park, Kyong Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; Van Balkom, Bas W M; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E.; Buée, Luc; Buzás, Edit I.; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S.; Desiderio, Dominic M.; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M.; Gardiner, Chris; Giebel, Bernd; Greening, David W.; Christina Gross, Julia; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F.; Hill, Michelle M.; Nolte-'T Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V.; Jayachandran, Muthuvel; Jee, Young Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; Van Leeuwen, Johannes; Lener, Thomas; Liu, Ming Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, Francois; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N.; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; Del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I.; Rodrigues, Marcio L.; Roh, Tae Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J.; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stepień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J.; Wai, Sun Nyunt; Witwer, Kenneth; Yánez-Mó, Maria; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

    2015-01-01

    Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We

  14. Understanding crumpling lipid vesicles at the gel phase transition

    Science.gov (United States)

    Hirst, Linda; Ossowski, Adam; Fraser, Matthew

    2011-03-01

    Wrinkling and crumpling transitions in different membrane types have been studied extensively in recent years both theoretically and computationally. There has also been very interesting recent work on defects in liquid crystalline shells. Lipid bilayer vesicles, widely used in biophysical research can be considered as a single layer smectic shell in the liquid crystalline phase. On cooling the lipid vesicle a transition to the gel phase may take place in which the lipid chains tilt and assume a more ordered packing arrangement. We observe large scale morphological changes in vesicles close to this transition point using fluorescence microscopy and investigate the possible mechanisms for this transition. Confocal microscopy is used to map 3D vesicle shape and crumpling length-scales. We also employ the molecular tilt sensitive dye, Laurdan to investigate the role of tilt domain formation on macroscopic structure. Funded by NSF CAREER award (DMR - BMAT #0852791).

  15. ABC triblock copolymer vesicles with mesh-like morphology.

    Science.gov (United States)

    Zhao, Wei; Chen, Dian; Hu, Yunxia; Grason, Gregory M; Russell, Thomas P

    2011-01-25

    Polymer vesicles made from poly(isoprene-b-styrene-b-2-vinyl pyridine) (PI-b-PS-b-P2VP) triblock copolymer confined within the nanopores of an anodic aluminum oxide (AAO) membrane are studied. It was found that these vesicles have well-defined, nanoscopic size, and complex microphase-separated hydrophobic membranes, comprised of the PS and PI blocks, while the coronas are formed by the P2VP block. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the membrane at a well-defined composition of the three blocks that can be tuned by changing the copolymer composition. The nanoscale confinement, copolymer composition, and subtle molecular interactions contribute to the generation of these vesicles with such unusual morphologies.

  16. Plasma membrane aquaporins mediates vesicle stability in broccoli.

    Directory of Open Access Journals (Sweden)

    Maria Del Carmen Martínez-Ballesta

    Full Text Available The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability.

  17. Assembly of cells and vesicles for organ engineering

    International Nuclear Information System (INIS)

    Taguchi, Tetsushi

    2011-01-01

    The development of materials and technologies for the assembly of cells and/or vesicles is a key for the next generation of tissue engineering. Since the introduction of the tissue engineering concept in 1993, various types of scaffolds have been developed for the regeneration of connective tissues in vitro and in vivo. Cartilage, bone and skin have been successfully regenerated in vitro, and these regenerated tissues have been applied clinically. However, organs such as the liver and pancreas constitute numerous cell types, contain small amounts of extracellular matrix, and are highly vascularized. Therefore, organ engineering will require the assembly of cells and/or vesicles. In particular, adhesion between cells/vesicles will be required for regeneration of organs in vitro. This review introduces and discusses the key technologies and materials for the assembly of cells/vesicles for organ regeneration. (topical review)

  18. 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.

  19. Extracellular vesicles in Alzheimer's disease: friends or foes? Focus on aβ-vesicle interaction.

    Science.gov (United States)

    Joshi, Pooja; Benussi, Luisa; Furlan, Roberto; Ghidoni, Roberta; Verderio, Claudia

    2015-03-03

    The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer's disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific molecules which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathology.

  20. CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation*

    OpenAIRE

    Nojiri, Mari; Loyet, Kelly M.; Klenchin, Vadim A.; Kabachinski, Gregory; Martin, Thomas F. J.

    2009-01-01

    CAPS (Ca2+-dependent activator protein for secretion) functions in priming Ca2+-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca2+-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in...

  1. Cystadenoma of the seminal vesicle. A case report

    DEFF Research Database (Denmark)

    Lundhus, E; Bundgaard, N; Sørensen, Flemming Brandt

    1984-01-01

    Cystadenomas of the seminal vesicle are extremely rare benign tumours, which only have been reported seven times earlier in the literature. The first Danish case is reported with discussion of symptomatology, pathology and treatment.......Cystadenomas of the seminal vesicle are extremely rare benign tumours, which only have been reported seven times earlier in the literature. The first Danish case is reported with discussion of symptomatology, pathology and treatment....

  2. Interaction and rheology of vesicle suspensions in confined shear flow

    Science.gov (United States)

    Shen, Zaiyi; Farutin, Alexander; Thiébaud, Marine; Misbah, Chaouqi

    2017-10-01

    Dynamics and rheology of a confined suspension of vesicles (a model for red blood cells) are studied numerically in two dimensions by using an immersed boundary lattice Boltzmann method. We pay particular attention to the link between the spatiotemporal organization and the rheology of the suspension. Besides confinement, we analyze the effect of concentration of the suspension, ϕ (defined as the area fraction occupied by the vesicles in the simulation domain), as well as the viscosity contrast λ (defined as the ratio between the viscosity of the fluid inside the vesicles, ηint, and that of the suspending fluid, ηext). The hydrodynamic interaction between two vesicles is shown to play a key role in determining the spatial organization. For λ =1 , the pair of vesicles settles into an equilibrium state with constant interdistance, which is regulated by the confinement. The equilibrium interdistance increases with the gap between walls, following a linear relationship. However, no stable equilibrium interdistance between two tumbling vesicles is observed for λ =10 . A quite ordered suspension is observed concomitant with the existence of an equilibrium interdistance between a vesicle pair. However, a disordered suspension prevails when no pair equilibrium interdistance exists, as occurs for tumbling vesicles. We then analyze the rheology, focusing on the effective viscosity, denoted as η , as well as on normalized viscosity, defined as [η ] =(η -ηext) /(ηextϕ ) . Ordering of the suspension is accompanied by a nonmonotonic behavior of [η ] with ϕ , while η exhibits plateaus. The nonmonotonic behavior of [η ] is suppressed when a disordered pattern prevails.

  3. Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

    Directory of Open Access Journals (Sweden)

    Vladislav M. Chernov

    2012-01-01

    Full Text Available For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity.

  4. Active elastohydrodynamics of vesicles in narrow blind constrictions

    Science.gov (United States)

    Fai, T. G.; Kusters, R.; Harting, J.; Rycroft, C. H.; Mahadevan, L.

    2017-11-01

    Fluid-resistance limited transport of vesicles through narrow constrictions is a recurring theme in many biological and engineering applications. Inspired by the motor-driven movement of soft membrane-bound vesicles into closed neuronal dendritic spines, here we study this problem using a combination of passive three-dimensional simulations and a simplified semianalytical theory for the active transport of vesicles forced through constrictions by molecular motors. We show that the motion of these objects is characterized by two dimensionless quantities related to the geometry and to the strength of forcing relative to the vesicle elasticity. We use numerical simulations to characterize the transit time for a vesicle forced by fluid pressure through a constriction in a channel and find that relative to an open channel, transport into a blind end leads to the formation of a smaller forward-flowing lubrication layer that strongly impedes motion. When the fluid pressure forcing is complemented by forces due to molecular motors that are responsible for vesicle trafficking into dendritic spines, we find that the competition between motor forcing and fluid drag results in multistable dynamics reminiscent of the real system. Our study highlights the role of nonlocal hydrodynamic effects in determining the kinetics of vesicular transport in constricted geometries.

  5. Bubble-induced microstreaming: guiding and destroying lipid vesicles

    Science.gov (United States)

    Marmottant, Philippe; Hilgenfeldt, Sascha

    2002-11-01

    Micron-sized bubbles respond with strong oscillations when submitted to ultrasound. This has led to their use as echographic contrast enhancers. The large energy and force densities generated by the collapsing bubbles also make them non-invasive mechanical tools: Recently, it has been reported that the interaction of cavitating bubbles with nearby cells can render the latter permeable to large molecules (sonoporation), suggesting prospects for drug delivery and gene transfection. We have developed a laboratory setup that allows for a controlled study of the interaction of single microbubbles with single lipid bilayer vesicles. Substituting vesicles for cell membranes is advantageous because the mechanical properties of vesicles are well-known. Microscopic observations reveal that vesicles near a bubble follow the vivid streaming motion set up by the bubble. The vesicles "bounce" off the bubble, being periodically accelerated towards and away from it, and undergo well-defined shape deformations along their trajectory in accordance with fluid-dynamical theory. Break-up of vesicles could also be observed.

  6. Identification of retrograde transport vesicles containing nerve growth factor in vivo

    International Nuclear Information System (INIS)

    Weible, M.W.; Sandow, S.L.; Ozsarac, N.; Hendry, I.A.; Grimes, M.L.

    2002-01-01

    Full text: Survival, differentiation, and development of responsive neurons are regulated by neurotrophins secreted from the target cells that they innervate. These responsive neurons must meet the challenge of transporting the neurotrophins chemical message from the target tissue to the soma; the distance of which may be a few millimetres to many meters. One hypothesis involves the formation of a signalling organelle at the neurite tip and subsequent retrograde axonal transport to the soma. This signalling vesicle is derived from the endocytosis of the neurotrophin-receptor complex. By modifying a method developed to isolate signalling endosomes from PC12 cells, we are able to isolate signalling vesicles from rat and mouse sciatic tissue. Approximately, 4 mole of I 125 -labelled neurotrophin was injected into the rodent foot pad and the sciatic nerve ligated under 88 μ/g ketamine and 16 μ/g rompun (i.p.) anaesthetic. All experiments had the approval of the ANU animal ethics committee. We achieved a recovery of 23% and 34% in the mouse and rat respectively of total transported iodinated neurotrophin accumulating on the distal side of the ligation. The homogenized tissue was characterized via differential centrifugation, blotted, and probed using antibodies to the neurotrophin receptors. Electron microscopy confirmed that the membrane pellet containing the transported neurotrophin from this in vivo preparation contained vesicular structures. Copyright (2002) Australian Neuroscience Society

  7. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

    Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

  8. Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    Nunzio Iraci

    2016-02-01

    Full Text Available Extracellular vesicles (EVs are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

  9. Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin‐Stimulated Glucose Uptake

    Science.gov (United States)

    Mleczko, Justyna; Ortega, Francisco J.; Falcon‐Perez, Juan Manuel; Wabitsch, Martin; Fernandez‐Real, Jose Manuel

    2018-01-01

    Scope We investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness. Methods and results EVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin‐stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin‐stimulated 2‐deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin‐mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2‐deoxyglucose uptake in adipocytes (p = 0.0159). Conclusion EVs released by stressed adipocytes impair insulin action in neighboring adipocytes. PMID:29292863

  10. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

    Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

  11. Seminal vesicle involvement at salvage radical prostatectomy.

    Science.gov (United States)

    Meeks, Joshua J; Walker, Marc; Bernstein, Melanie; Eastham, James A

    2013-06-01

    To describe the incidence and clinical outcomes of seminal vesicle invasion (SVI) at salvage radical prostatectomy (SRP) and to describe the accuracy of SV biopsy. As SRP is used after biochemical recurrence (BCR) of prostate cancer after radiotherapy (RT) to gain local oncological control. The SVs receive lower doses of radiation from external-beam RT (EBRT) to avoid rectal exposure and are not targeted with brachytherapy (BT) with low-risk prostate cancer. SRP was performed on 206 men with BCR after RT at a tertiary care institution between 1998 and 2011. Post-RT biopsy and SRP specimens were reviewed by a genitourinary pathologist. SVI was detected in 65 (32%) of 206 patients. No difference was found between EBRT alone (65% vs 63%) and BT (29% vs 31%) with or without EBRT in patients with SVI. Men with SVI had higher rates of cT3 disease (20% vs 8%) and Gleason score ≥ 8 at SRP (52% vs 21%). BCR-free survival at 5 years was 18% and 56% in patients with and without SVI (hazard ratio 2.85, 95% confidence interval 1.87-4.36, P < 0.001), yet the rate of local recurrence was low (11%). Prostate cancer was identified in nine of 18 patients who underwent SV biopsy and was the only location of prostate cancer in two patients. SVI is a prognostic indicator for BCR after SRP, but local recurrence in patients with SVI after SRP remains low. We recommend SV biopsy to improve staging and cancer detection in men with BCR after radiotherapy. © 2013 BJU International.

  12. Analysis of long-chain fatty acid binding activity in vesicles of the outer membrane generated from Escherchia coli

    International Nuclear Information System (INIS)

    Black, P.N.

    1987-01-01

    Escherichia coli transports long-chain fatty acids across the dual membrane by a high affinity, saturable, energy-dependent process. The fadL gene codes for an outer membrane protein which appears to act specifically as a long-chain fatty acid binding protein when fatty acid utilization is blocked by mutation. In an effort to understand the function of the fadL gene product, FLP, membranes have been isolated from fadL + and fadL - strains following osmotic lysis. Following isolation, total membranes were separated into inner and outer membrane fractions and assayed for long-chain fatty acid binding activity. Outer membrane vesicles were incubated 2-5 min at 37 0 C with 3 H oleate (C/sub 18:1/), cooled to 0 0 C, and centrifuged through a Lipidex 100 column for 3 min to remove the unbound fatty acid. The level of fatty acid binding was quantitated by scintillation counting of the eluate. Outer membrane vesicles generated from a fadL + strain bind 325 pmol fatty acid/mg protein whereas vesicles generated for a mutant strain bind 175 pmol fatty acid/mg protein. These data suggest that FLP acts at least as a long-chain fatty acid binding protein on the surface of the cell

  13. 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

  14. Insight into the template effect of vesicles on the laccase-catalyzed oligomerization of N-phenyl-1,4-phenylenediamine from Raman spectroscopy and cyclic voltammetry measurements

    Science.gov (United States)

    Ležaić, Aleksandra Janoševic; Luginbühl, Sandra; Bajuk-Bogdanović, Danica; Pašti, Igor; Kissner, Reinhard; Rakvin, Boris; Walde, Peter; Ćirić-Marjanović, Gordana

    2016-08-01

    We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with Trametes versicolor laccase and dissolved O2 in the presence of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) vesicles (80-100 nm diameter) as templates. The conversion of PADPA into oligomeric products, poly(PADPA), was monitored during the reaction by in situ Raman spectroscopy. The results obtained are compared with UV/vis/NIR and EPR measurements. All three complementary methods indicate that at least some of the poly(PADPA) products, formed in the presence of AOT vesicles, resemble the conductive emeraldine salt form of polyaniline (PANI-ES). The Raman measurements also show that structural units different from those of “ordinary” PANI-ES are present too. Without vesicles PANI-ES-like products are not obtained. For the first time, the as-prepared stable poly(PADPA)-AOT vesicle suspension was used directly to coat electrodes (without product isolation) for investigating redox activities of poly(PADPA) by cyclic voltammetry (CV). CV showed that poly(PADPA) produced with vesicles is redox active not only at pH 1.1-as expected for PANI-ES-but also at pH 6.0, unlike PANI-ES and poly(PADPA) synthesized without vesicles. This extended pH range of the redox activity of poly(PADPA) is important for applications.

  15. SENSITIVE EFFECTS OF POTASSIUM AND CALCIUM CHANNEL BLOCKING AND ATP-SENSITIVE POTASSIUM CHANNEL ACTIVATORS ON SEMINAL VESICLE SMOOTH MUSCLE CONTRACTIONS

    Directory of Open Access Journals (Sweden)

    H SADRAEI

    2000-12-01

    Full Text Available Background. Seminal vesicle smooth muscle contraction is mediated through sympathetic and parasympathetic neurons activity. Although seminal vesicle plays an important role in male fertility, but little attention is given to mechanism involved in contraction of this organ.
    Methods. In this study effects of drugs which activate ATP - sensitive K channels and blockers of K and Ca channels were examined on contraction of guinea - pig isolated seminal vesicle due to electrical filled stimulation (EFS, noradrenaline, carbachol and KCI.
    Results. The K channel blocker tetraethyl ammonium potentate the EFS responses at all frequencies, while, the ATP - sensitive K channel inhibitor glibenclamide and the K channel opener levcromakalim, diazoxide, minoxidil and Ca channel blocker nifedipine all had relaxant effect on guinea - pig seminal vesicle.
    Discussion. This study indicate that activities of K and Ca channels is important in regulation of seminal vesicle contraction due to nerve stimulation, noradrenaline or carbachol.

  16. Overall energy conversion efficiency of a photosynthetic vesicle

    Energy Technology Data Exchange (ETDEWEB)

    Sener, Melih [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Strumpfer, Johan [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States; Singharoy, Abhishek [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Hunter, C. Neil [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom; Schulten, Klaus [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States

    2016-08-26

    The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

  17. Adhesion signals of phospholipid vesicles at an electrified interface.

    Science.gov (United States)

    DeNardis, Nadica Ivošević; Žutić, Vera; Svetličić, Vesna; Frkanec, Ruža

    2012-09-01

    General adhesion behavior of phospholipid vesicles was examined in a wide range of potentials at the mercury electrode by recording time-resolved adhesion signals. It was demonstrated that adhesion-based detection is sensitive to polar headgroups in phospholipid vesicles. We identified a narrow potential window around the point of zero charge of the electrode where the interaction of polar headgroups of phosphatidylcholine vesicles with the substrate is manifested in the form of bidirectional signals. The bidirectional signal is composed of the charge flow due to the nonspecific interaction of vesicle adhesion and spreading and of the charge flow due to a specific interaction of the negatively charged electrode and the most exposed positively charged choline headgroups. These signals are expected to appear only when the electrode surface charge density is less than the surface charge density of the choline groups at the contact interface. In comparison, for the negatively charged phosphatidylserine vesicles, we identified the potential window at the mercury electrode where charge compensation takes place, and bidirectional signals were not detected.

  18. Models for randomly distributed nanoscopic domains on spherical vesicles

    Science.gov (United States)

    Anghel, Vinicius N. P.; Bolmatov, Dima; Katsaras, John

    2018-06-01

    The existence of lipid domains in the plasma membrane of biological systems has proven controversial, primarily due to their nanoscopic size—a length scale difficult to interrogate with most commonly used experimental techniques. Scattering techniques have recently proven capable of studying nanoscopic lipid domains populating spherical vesicles. However, the development of analytical methods able of predicting and analyzing domain pair correlations from such experiments has not kept pace. Here, we developed models for the random distribution of monodisperse, circular nanoscopic domains averaged on the surface of a spherical vesicle. Specifically, the models take into account (i) intradomain correlations corresponding to form factors and interdomain correlations corresponding to pair distribution functions, and (ii) the analytical computation of interdomain correlations for cases of two and three domains on a spherical vesicle. In the case of more than three domains, these correlations are treated either by Monte Carlo simulations or by spherical analogs of the Ornstein-Zernike and Percus-Yevick (PY) equations. Importantly, the spherical analog of the PY equation works best in the case of nanoscopic size domains, a length scale that is mostly inaccessible by experimental approaches such as, for example, fluorescent techniques and optical microscopies. The analytical form factors and structure factors of nanoscopic domains populating a spherical vesicle provide a new and important framework for the quantitative analysis of experimental data from commonly studied phase-separated vesicles used in a wide range of biophysical studies.

  19. A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing

    Directory of Open Access Journals (Sweden)

    Robert Vogel

    2016-09-01

    Full Text Available Background: Understanding the pathogenic role of extracellular vesicles (EVs in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. Materials and Methods: A standardized methodology to measure the concentration of extracellular vesicles (EVs has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV and consecutively measured with tunable resistive pulse sensing (TRPS. Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets. Results: PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV and EV (filtered with qEV concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs. Conclusion: The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

  20. Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

    International Nuclear Information System (INIS)

    Mulvey, Hillary E.; Chang, Audrey; Adler, Jason; Del Tatto, Michael; Perez, Kimberly; Quesenberry, Peter J.; Chatterjee, Devasis

    2015-01-01

    Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer

  1. 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.

  2. Irregular bilayer structure in vesicles prepared from Halobacterium cutirubrum lipids

    Science.gov (United States)

    Lanyi, J. K.

    1974-01-01

    Fluorescent probes were used to study the structure of the cell envelope of Halobacterium cutirubrum, and, in particular, to explore the effect of the heterogeneity of the lipids in this organism on the structure of the bilayers. The fluorescence polarization of perylene was followed in vesicles of unfractionated lipids and polar lipids as a function of temperature in 3.4 M solutions of NaCl, NaNO3, and KSCN, and it was found that vesicles of unfractionated lipids were more perturbed by chaotropic agents than polar lipids. The dependence of the relaxation times of perylene on temperature was studied in cell envelopes and in vesicles prepared from polar lipids, unfractionated lipids, and mixtures of polar and neutral lipids.

  3. Vesicle biomechanics in a time-varying magnetic field.

    Science.gov (United States)

    Ye, Hui; Curcuru, Austen

    2015-01-01

    Cells exhibit distortion when exposed to a strong electric field, suggesting that the field imposes control over cellular biomechanics. Closed pure lipid bilayer membranes (vesicles) have been widely used for the experimental and theoretical studies of cellular biomechanics under this electrodeformation. An alternative method used to generate an electric field is by electromagnetic induction with a time-varying magnetic field. References reporting the magnetic control of cellular mechanics have recently emerged. However, theoretical analysis of the cellular mechanics under a time-varying magnetic field is inadequate. We developed an analytical theory to investigate the biomechanics of a modeled vesicle under a time-varying magnetic field. Following previous publications and to simplify the calculation, this model treated the inner and suspending media as lossy dielectrics, the membrane thickness set at zero, and the electric resistance of the membrane assumed to be negligible. This work provided the first analytical solutions for the surface charges, electric field, radial pressure, overall translational forces, and rotational torques introduced on a vesicle by the time-varying magnetic field. Frequency responses of these measures were analyzed, particularly the frequency used clinically by transcranial magnetic stimulation (TMS). The induced surface charges interacted with the electric field to produce a biomechanical impact upon the vesicle. The distribution of the induced surface charges depended on the orientation of the coil and field frequency. The densities of these charges were trivial at low frequency ranges, but significant at high frequency ranges. The direction of the radial force on the vesicle was dependent on the conductivity ratio between the vesicle and the medium. At relatively low frequencies (biomechanics under a time-varying magnetic field. Biological effects of clinical TMS are not likely to occur via alteration of the biomechanics of brain

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    The neurodegenerative illness Familial Danish Dementia (FDD) is linked to formation and aggregation of the 34-residue ADan peptide, whose cytotoxicity may be mediated by membrane interactions. Here we characterize the derived peptide SerADan, in which the two cysteines found in ADan have been....... 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...

  5. Lipids, lipid bilayers and vesicles as seen by neutrons

    International Nuclear Information System (INIS)

    Seto, Hideki

    2011-01-01

    Lipid molecules self-assemble into bilayers in water with their hydrocarbon chains facing inward due to their amphiphilic nature. The structural and dynamical properties of lipids and lipid bilayers have been studied by neutron scattering intensively. In this article, 3 topics are shown as typical examples. 1) a time-resolved small-angle neutron scattering on uni-lamellar vesicles composed of deuterated and protonated lipids to determine lipid kinetics, 2) small-angle neutron scattering to investigate spontaneous formation of nanopores on uni-lamellar vesicles, and 3) neutron spin echo study to determine bending modulus of lipid bilayers. (author)

  6. Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular VesiclesSummary

    Directory of Open Access Journals (Sweden)

    Takahiro Sanada

    2017-03-01

    Full Text Available Background & Aims: An extracellular vesicle (EV is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV infection. Methods: We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells. Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy. Results: Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA–transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA–transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies. Conclusions: These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization–resistant route of HBV infection. Keywords: HBV, Extracellular Vesicles, Transmission Pathway

  7. Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

    Directory of Open Access Journals (Sweden)

    Michael H. Norris

    2018-01-01

    Full Text Available Burkholderia mallei is a host-adapted Gram-negative mammalian pathogen that causes the severe disease glanders. Glanders can manifest as a rapid acute progression or a chronic debilitating syndrome primarily affecting solipeds and humans in close association with infected animals. In USA, B. mallei is classified as one of the most important bacterial biothreat agents. Presently, there is no licensed glanders vaccine available for humans or animals. In this work, outer membrane vesicles (OMVs were isolated from three attenuated biosafe bacterial strains, Burkholderia pseudomallei Bp82, B. thailandensis E555, and B. thailandensis TxDOH and used to vaccinate mice. B. thailandensis OMVs induced significantly higher antibody responses that were investigated. B. mallei specific serum antibody responses were of higher magnitude in mice vaccinated with B. thailandensis OMVs compared to levels in mice vaccinated with B. pseudomallei OMVs. OMVs derived from biosafe strains protected mice from acute lethal glanders with vesicles from the two B. thailandensis strains affording significant protection (>90% up to 35 days post-infection with some up to 60 days. Organ loads from 35-day survivors indicated bacteria colonization of the lungs, liver, and spleen while those from 60 days had high CFUs in the spleens. The highest antibody producing vaccine (B. thailandensis E555 OMVs also protected C57BL/6 mice from acute inhalational glanders with evidence of full protection.

  8. Vesicle interactions with polyamino acids and antibody: in vitro and in vivo studies

    International Nuclear Information System (INIS)

    Dunnick, J.K.; McDougall, I.R.; Aragon, S.; Goris, M.L.; Kriss, J.P.

    1975-01-01

    Artificial spherules or vesicles of 900 A in diameter formed from phosphatidylcholine and gangliosides and enclosing /sup 99m/TcO 4 - (standard preparation) survive intact in the circulation of the mouse. Polyamino acids and protein have been incorporated into and onto the vesicles; such vesicles remain intact as determined by diffusion dialysis studies and by electron paramagnetic resonance studies of vesicles enclosing spin label. In studying the distribution of polyamino acid-vesicles and protein vesicles in vivo, it was found that the latter distribute differently from standard vesicles or free protein alone whereas aromatic polyamino acid-vesicles concentrate in the liver and spleen to a greater extent than standard vesicles. The permeability and stability characteristics of vesicles may be preserved when they are modified by the addition of protein or polyamino acids and that such modification of vesicles may be associated with an alteration of their fate in vivo. The potential exists to use vesicles as carriers of radiopharmaceuticals and other drugs and to direct the vesicles preferentially to tissue targets in vivo. (U.S.)

  9. Primary vesicles, vesicle-rich segregation structures and recognition of primary and secondary porosities in lava flows from the Paraná igneous province, southern Brazil

    Science.gov (United States)

    Barreto, Carla Joana S.; de Lima, Evandro F.; Goldberg, Karin

    2017-04-01

    This study focuses on a volcanic succession of pāhoehoe to rubbly lavas of the Paraná-Etendeka Province exposed in a single road profile in southernmost Brazil. This work provides an integrated approach for examining primary vesicles and vesicle-rich segregation structures at the mesoscopic scale. In addition, this study provides a quantitative analysis of pore types in thin section. We documented distinct distribution patterns of vesicle and vesicle-rich segregation structures according to lava thickness. In compound pāhoehoe lavas, the cooling allows only vesicles (pipe vesicles to be frozen into place. In inflated pāhoehoe lavas, vesicles of different sizes are common, including pipe vesicles, and also segregation structures such as proto-cylinders, cylinders, cylinder sheets, vesicle sheets, and pods. In rubbly lavas, only vesicles of varying sizes occur. Gas release from melt caused the formation of primary porosity, while hydrothermal alteration and tectonic fracturing are the main processes that generated secondary porosity. Although several forms of porosity were created in the basaltic lava flows, the precipitation of secondary minerals within the pores has tended to reduce the original porosities. Late-stage fractures could create efficient channel networks for possible hydrocarbon/groundwater migration and entrapment owing to their ability to connect single pores. Quantitative permeability data should be gathered in future studies to confirm the potential of these lavas for store hydrocarbons or groundwater.

  10. Outer Membrane Vesicles From Probiotic and Commensal Escherichia coli Activate NOD1-Mediated Immune Responses in Intestinal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    María-Alexandra Cañas

    2018-03-01

    Full Text Available Gut microbiota plays a critical role in maintaining human intestinal homeostasis and host health. Bacterial extracellular vesicles are key players in bacteria–host communication, as they allow delivery of effector molecules into the host cells. Outer membrane vesicles (OMVs released by Gram-negative bacteria carry many ligands of pattern recognition receptors that are key components of innate immunity. NOD1 and NOD2 cytosolic receptors specifically recognize peptidoglycans present within the bacterial cell wall. These intracellular immune receptors are essential in host defense against bacterial infections and in the regulation of inflammatory responses. Recent contributions show that NODs are also fundamental to maintain intestinal homeostasis and microbiota balance. Peptidoglycan from non-invasive pathogens is delivered to cytosolic NODs through OMVs, which are internalized via endocytosis. Whether this pathway could be used by microbiota to activate NOD receptors remains unexplored. Here, we report that OMVs isolated from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 activate NOD1 signaling pathways in intestinal epithelial cells. NOD1 silencing and RIP2 inhibition significantly abolished OMV-mediated activation of NF-κB and subsequent IL-6 and IL-8 expression. Confocal fluorescence microscopy analysis confirmed that endocytosed OMVs colocalize with NOD1, trigger the formation of NOD1 aggregates, and promote NOD1 association with early endosomes. This study shows for the first time the activation of NOD1-signaling pathways by extracellular vesicles released by gut microbiota.

  11. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

    International Nuclear Information System (INIS)

    Chase, H.S. Jr.; Al-Awqati, Q.

    1983-01-01

    Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself

  12. MRI, CT and TRUS imaging of seminal vesicle metastasis

    International Nuclear Information System (INIS)

    Larsson, P.; Blomqvist, L.; Norming, U.

    1997-01-01

    We present a case of a testicular germ-cell metastasis in the seminal vesicle. Diagnostic imaging with transrectal ultrasonography (TRUS), CT, and MRI was performed. This case emphasizes the role of MRI in the evaluation of patients with pathology in the pelvic region. (orig.)

  13. Glycosylation of extracellular vesicles : current knowledge, tools and clinical perspectives

    NARCIS (Netherlands)

    Williams, Charles; Royo, Felix; Aizpurua-Olaizola, Oier; Pazos, Raquel; Boons, Geert-Jan; Reichardt, Niels-Christian; Falcon-Perez, Juan M

    2018-01-01

    It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed,

  14. Continuous fabrication of polymeric vesicles and nanotubes with fluidic channe

    NARCIS (Netherlands)

    Peng, F.; Deng, N.-N.; Tu, Y.; van Hest, J.C.M.; Wilson, D.A.

    2017-01-01

    Fluidic channels were employed to induce the self-assembly of poly(ethylene glycol)-b-polystyrene into polymeric vesicles and nanotubes. The laminar flow in the device enables controlled diffusion of two miscible liquids at the phase boundary, leading to the formation of homogeneous polymeric

  15. Primary malignancy of seminal vesicle: A rare entity

    Directory of Open Access Journals (Sweden)

    Rajaraman Ramamurthy

    2011-01-01

    Full Text Available We report a rare case of seminal vesicle malignancy (primitive neuro ectodermal tumor in a 40-year-old male patient. He was treated with enbloc resection of the tumor and ureteric reimplantation. In view of the rarity of this entity, management of these tumors should be individualized.

  16. Dynamics of Shape Fluctuations of Quasi-spherical Vesicles Revisited

    DEFF Research Database (Denmark)

    Miao, L.; Lomholt, Michael Andersen; Kleis, J.

    2002-01-01

    In this paper, the dynamics of spontaneous shape fluctuations of a single, giant quasi-spherical vesicle formed from a single lipid species is revisited theoretically. A coherent physical theory for the dynamics is developed based on a number of fundamental principles and considerations, and a sy......In this paper, the dynamics of spontaneous shape fluctuations of a single, giant quasi-spherical vesicle formed from a single lipid species is revisited theoretically. A coherent physical theory for the dynamics is developed based on a number of fundamental principles and considerations...... of the phenomenological constants in a canonical continuum description of fluid lipid-bilayer membranes and shown the consequences of this new interpretation in terms of the characteristics of the dynamics of vesicle shape fluctuations. Moreover, we have used the systematic formulation of our theory as a framework...... against which we have discussed the previously existing theories and their discrepancies. Finally, we have made a systematic prediction about the system-dependent characteristics of the relaxation dynamics of shape fluctuations of quasi-spherical vesicles with a view of experimental studies...

  17. Seminal vesicle abscess causing unilateral hydroureteronephrosis: A case report

    Directory of Open Access Journals (Sweden)

    Vittorio Imperatore

    2017-12-01

    Full Text Available Seminal vesicle abscess (SVA is a rare urologic entity. It mainly occurs in subjects with predisposing factors and may be associated with other urogenital infections. We describe the case of a diabetic subject with SVA associated with funiculitis, epididymitis and obstructive pyelonephritis. Treatment consisted of laparotomic surgical drainage of the abscess and ureteral stent placement.

  18. A Pathogenic Potential of Acinetobacter baumannii-Derived Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    Jong Suk Jin

    2011-12-01

    Full Text Available Acinetobacter baumannii secretes outer membrane vesicles (OMVs. A. baumannii OMVs deliver many virulence factors to host cells and then induce cytotoxicity and innate immune response. OMVs secreted from bacteria contribute directly to host pathology during A. baumannii infection.

  19. Penetration and fusion of phospholipid vesicles by lysozyme

    International Nuclear Information System (INIS)

    Kim, J.; Kim, H.

    1989-01-01

    The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([ 125 I]iodophenyl)diazirine ([ 125 I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [ 125 I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion

  20. Penetration and fusion of phospholipid vesicles by lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Kim, J.; Kim, H. (Korea Advanced Institute of Science and Technology, Seoul)

    1989-10-01

    The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-(({sup 125}I)iodophenyl)diazirine (({sup 125}I)TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent ({sup 125}I)TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.

  1. Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

    DEFF Research Database (Denmark)

    Goettsch, Claudia; Hutscheson, JD; Aikawa, M

    2016-01-01

    obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin...

  2. Ultrasound-guided seminal vesicle biopsies in prostate cancer

    NARCIS (Netherlands)

    Wymenga, LFA; Duisterwinkel, FJ; Groenier, K; Mensink, HJA

    2000-01-01

    Invasion of prostatic adenocarcinoma into the seminal vesicles (SV) is generally accepted as an index of poor prognosis. The pre-operative identification of SV invasion is an important element in staging since it may alter subsequent treatment decisions. We studied the possibility of diagnosing SV

  3. Transcutol containing vesicles for topical delivery of minoxidil.

    Science.gov (United States)

    Mura, Simona; Manconi, Maria; Valenti, Donatella; Sinico, Chiara; Vila, Amparo Ofelia; Fadda, Anna Maria

    2011-04-01

    The aim of this work was to evaluate the ability of Transcutol (Trc) to produce elastic vesicles with soy lecithin (SL) and study the influence of the obtained vesicles on in vitro (trans)dermal delivery of minoxidil. To this purpose, so-called penetration enhancer-containing vesicles (PEVs) were prepared using Trc aqueous solutions (5-10-20-30% v/v) as hydrophilic phase. SL liposomes, without Trc, were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, deformability, and rheological behavior. The influence of the obtained PEVs on (trans)dermal delivery of minoxidil was studied by in vitro diffusion experiments through pig skin. Results showed that all prepared PEVs were able to give good entrapment efficiency (E%≈67) similar to that of conventional liposomes. Trc-containing PEVs showed to be more deformable than liposomes only when minoxidil was loaded in 5 and 10% Trc-containing vesicles. Rheological studies showed that PEVs have higher fluidity than conventional liposomes. All PEVs showed a higher stability than liposomes as shown by studying zeta potential and size distribution during three months. Results of in vitro diffusion experiments showed that Trc-containing PEVs are able to deliver minoxidil to deep skin layers without any transdermal permeation.

  4. Inflammation leads to distinct populations of extracellular vesicles from microglia

    DEFF Research Database (Denmark)

    Yang, Yiyi; Boza-Serrano, Antonio; Dunning, Christopher J.R.

    2018-01-01

    Background: Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication...

  5. Intermedin inhibits norepinephrine-induced contraction of rat seminal vesicle

    Directory of Open Access Journals (Sweden)

    P.F. Wong

    2014-09-01

    Conclusion: The results demonstrated that the inhibitory action of IMD on NE-induced seminal vesicle contraction was mediated via the ADM receptor(s and the nitric oxide production pathway, partially by the IMD receptor, but not by the CGRP receptor and the cAMP-PKA pathway.

  6. Calcium transport in vesicles energized by cytochrome oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Rosier, Randy N. [Univ. of Rochester, NY (United States)

    1979-01-01

    Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K+ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K+ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

  7. Thin film drainage between pre-inflated capsules or vesicles

    Science.gov (United States)

    Keh, Martin; Walter, Johann; Leal, Gary

    2013-11-01

    Capsules and vesicles are often used as vehicles to carry active ingredients or fragrance in drug delivery and consumer products and oftentimes in these applications the particles may be pre-inflated due to the existence of a small osmotic pressure difference between the interior and exterior fluid. We study the dynamics of thin film drainage between capsules and vesicles in flow as it is crucial to fusion and deposition of the particles and, therefore, the stability and effectiveness of the products. Simulations are conducted using a numerical model coupling the boundary integral method for the motion of the fluids and a finite element method for the membrane mechanics. For low capillary numbers, the drainage behavior of vesicles and capsules are approximately the same, and also similar to that of drops as the flow-independent and uniform tension due to pre-inflation dominates. The tension due to deformation caused by flow will become more important as the strength of the external flow (i.e. the capillary number) increases. In this case, the shapes of the thin film region are fundamentally different for capsules and vesicles, and the drainage behavior in both cases differs from a drop. Funded by P&G.

  8. Brivaracetam augments short-term depression and slows vesicle recycling.

    Science.gov (United States)

    Yang, Xiaofeng; Bognar, Joseph; He, Tianyu; Mohammed, Mouhari; Niespodziany, Isabelle; Wolff, Christian; Esguerra, Manuel; Rothman, Steven M; Dubinsky, Janet M

    2015-12-01

    Brivaracetam (BRV) decreases seizure activity in a number of epilepsy models and binds to the synaptic vesicle glycoprotein 2A (SV2A) with a higher affinity than the antiepileptic drug levetiracetam (LEV). Experiments were performed to determine if BRV acted similarly to LEV to induce or augment short-term depression (STD) under high-frequency neuronal stimulation and slow synaptic vesicle recycling. Electrophysiologic field excitatory postsynaptic potential (fEPSP) recordings were made from CA1 synapses in rat hippocampal slices loaded with BRV or LEV during intrinsic activity or with BRV actively loaded during hypertonic stimulation. STD was examined in response to 5 or 40 Hz stimulus trains. Presynaptic release of FM1-43 was visualized using two-photon microscopy to assess drug effects upon synaptic vesicle mobilization. When hippocampal slices were incubated in 0.1-30 μm BRV or 30 μm-1 mm LEV for 3 h, the relative CA1 field EPSPs decreased over the course of a high-frequency train of stimuli more than for control slices. This STD was frequency- and concentration-dependent, with BRV being 100-fold more potent than LEV. The extent of STD depended on the length of the incubation time for both drugs. Pretreatment with LEV occluded the effects of BRV. Repeated hypertonic sucrose treatments and train stimulation successfully unloaded BRV from recycling vesicles and reversed BRVs effects on STD, as previously reported for LEV. At their maximal concentrations, BRV slowed FM1-43 release to a greater extent than in slices loaded with LEV during prolonged stimulation. BRV, similar to LEV, entered into recycling synaptic vesicles and produced a frequency-dependent decrement of synaptic transmission at 100-fold lower concentrations than LEV. In addition, BRV slowed synaptic vesicle mobilization more effectively than LEV, suggesting that these drugs may modify multiple functions of the synaptic vesicle protein SV2A to curb synaptic transmission and limit epileptic activity

  9. Molecular Recognition of Vesicles : Host-Guest Interactions Combined with Specific Dimerization of Zwitterions

    NARCIS (Netherlands)

    Voskuhl, Jens; Fenske, Tassilo; Stuart, Marc C. A.; Wibbeling, Birgit; Schmuck, Carsten; Ravoo, Bart Jan

    2010-01-01

    The aggregation of beta-cyclodextrin vesicles can be induced by an adamantyl-substituted zwitterionic guanidiniocarbonylpyrrole carboxylate guest molecule (1). Upon addition of 1 to the cyclodextrin vesicles at neutral pH, the vesicles aggregate (but do not fuse), as shown by using UV/Vis and

  10. Pulsed-laser polymerization in compartmentalized liquids. 1. Polymerization in vesicles

    NARCIS (Netherlands)

    Jung, M.; Casteren, van I.A.; Monteiro, M.J.; Herk, van A.M.; German, A.L.

    2000-01-01

    Polymerization in vesicles is a novel type of polymerization in heterogeneous media, leading to parachute-like vesicle-polymer hybrid morphologies. To explore the kinetics of vesicle polymerizations and to learn more about the actual locus of polymerization we applied the pulsed-laser polymerization

  11. Seminal vesicle metastasis after partial hepatectomy for hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Gong, Li; Zheng, Minwen; Li, Yanhong; Zhang, Wendong; Bu, Wangjun; Shi, Lifang; Zhang, Wei; Yan, Hong

    2011-01-01

    Metastasis to the seminal vesicle is extremely rare for hepatocellular carcinoma (HCC). To our knowledge, it has been not reported in literature. The purpose of the present paper was to report a case of metastasis to the seminal vesicle after HCC resection, along with its histological features and immunohistochemical characteristics. A 46-year-old Chinese man was admitted to our hospital due to abdominal distension. He had a history of HCC related to hepatitis B virus infection. Moreover, left partial hepatectomy was performed in another hospital 28 months ago, and right partial hepatectomy for HCC recurrence in our hospital 4 months ago. After resection, radiofrequency ablation therapy had been performed. About 27 months after the initial operation, contrast-enhanced computed tomography (CT) of the pelvic cavity revealed a mass with homogeneous enhancement in the seminal vesicle. Transrectal needle biopsy revealed a poorly differentiated adenocarcinoma. Therefore, seminal vesiculectomy was resected. The histological diagnosis of the removed tumor was compatible with the original HCC. Immunohistochemical examination demonstrated that the tumor cells were positive for glypican-3 (GPC3), alpha-fetoprotein (AFP), hepatocyte paraffin-1 (Hep Par 1), cytokeratin 18 (CK 18), and hepatocyte antigen, which confirmed that the seminal vesicle tumor was a metastatic tumor of HCC. However, CT subsequently revealed multiple metastatic foci in the abdominal and pelvic cavities in May 2009 and August 2009, respectively. The seminal vesicle is an extremely rare metastatic site for HCC, and the prognosis is very poor. A combination of clinical and pathological features is necessary for a correct diagnosis, and primary tumor should be excluded before diagnosing metastatic foci

  12. Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

    Science.gov (United States)

    Gerstle, Zoe; Desai, Rohan; Veatch, Sarah L

    2018-01-01

    Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy. © 2018 Elsevier Inc. All rights reserved.

  13. Probing Interactions between AuNPs/AgNPs and Giant Unilamellar Vesicles (GUVs Using Hyperspectral Dark-field Microscopy

    Directory of Open Access Journals (Sweden)

    Anupama Bhat

    2018-03-01

    Full Text Available Noble metallic nanoparticles (NPs such as gold and silver nanoparticles (AuNPs and AgNPs have been shown to exhibit anti-tumor effect in anti-angiogenesis, photothermal and radio therapeutics. On the other hand, cell membranes are critical locales for specific targeting of cancerous cells. Therefore, NP-membrane interactions need be studied at molecular level to help better understand the underlying physicochemical mechanisms for future applications in cancer nanotechnology. Herein, we report our study on the interactions between citrate stabilized colloidal AuNPs/AgNPs (10 nm in size and giant unilamellar vesicles (GUVs using hyperspectral dark-field microscopy. GUVs are large model vesicle systems well established for the study of membrane dynamics. GUVs used in this study were prepared with dimyristoyl phosphatidylcholine (DMPC and doped with cholesterol at various molar concentrations. Both imaging and spectral results support that AuNPs and AgNPs interact very differently with GUVs, i.e., AuNPs tend to integrate in between the lipid bilayer and form a uniform golden-brown crust on vesicles, whereas AgNPs are bejeweled on the vesicle surface as isolated particles or clusters with much varied configurations. The more disruptive capability of AuNPs is hypothesized to be responsible for the formation of golden brown crusts in AuNP-GUV interaction. GUVs of 20 mol% CHOL:DMPC were found to be a most economical concentration for GUVs to achieve the best integrity and the least permeability, consistent with the finding from other phase studies of lipid mixture that the liquid-ordered domains have the largest area fraction of the entire membrane at around 20 mol% of cholesterol.

  14. Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens

    DEFF Research Database (Denmark)

    Pors, Susanne Elisabeth; Pedersen, Ida Just; Skjerning, Ragnhild Bager

    2016-01-01

    Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have...... ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re...

  15. Elastic vesicles for transdermal drug delivery of hydrophilic drugs: a comparison of important physicochemical characteristics of different vesicle types.

    Science.gov (United States)

    Ntimenou, Vassiliki; Fahr, Alfred; Antimisiaris, Sophia G

    2012-08-01

    The aim of this study is to evaluate the influence of different lipid vesicular systems on the skin permeation ability of hydrophilic molecules, and understand if and which vesicle physicochemical properties may be used as predictive tools. Calcein and carboxyfluorescein were used as hydrophilic drug models. All vesicles (conventional liposomes [CLs], transfersomes [TRs] and invasomes [INVs]), were characterized for particle size distribution, zeta-potential, vesicular shape and morphology, encapsulation efficiency, integrity, colloidal stability, elasticity and finally in vitro human skin permeation. Dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM) defined that almost all vesicles had spherical structure, low polydispersity (PI Elasticity values (measured by extrusion through membranes) were in the order INVs > TRs > CLs. Three vesicle types were selected (having different elasticity) and in vitro skin permeation experiments demonstrated that calcein permeation was minimal from an aqueous solution, slightly enhanced from CLs, and enhanced by 1.8 and 7.2 times from TRs and INVs, respectively. Permeation and elasticity values were correlated by rank order but not linearly, indicating that elasticity can be used as a crude predictive tool for enhancement of skin transport. Drug encapsulation efficiency was not found to be an important factor in the current study.

  16. Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

    Science.gov (United States)

    Bai, Jaewoo; Kim, Seul I; Ryu, Sangryeol

    2014-01-01

    Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes. PMID:24935973

  17. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.

    Science.gov (United States)

    Lötvall, Jan; Hill, Andrew F; Hochberg, Fred; Buzás, Edit I; Di Vizio, Dolores; Gardiner, Christopher; Gho, Yong Song; Kurochkin, Igor V; Mathivanan, Suresh; Quesenberry, Peter; Sahoo, Susmita; Tahara, Hidetoshi; Wauben, Marca H; Witwer, Kenneth W; Théry, Clotilde

    2014-01-01

    Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

  18. Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2016-02-01

    Full Text Available The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.

  19. Genetically controlled fusion, exocytosis and fission of artificial vesicles-a roadmap

    DEFF Research Database (Denmark)

    Bönzli, Eva; Hadorn, Maik; de Lucrezia, Davide

    2011-01-01

    were shown to fuse if a special class of viral proteins, termed fusogenic peptides, were added to the external medium (Nomura et al. 2004). 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...

  20. Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

    Science.gov (United States)

    Sunami, Takeshi; Caschera, Filippo; Morita, Yuuki; Toyota, Taro; Nishimura, Kazuya; Matsuura, Tomoaki; Suzuki, Hiroaki; Hanczyc, Martin M; Yomo, Tetsuya

    2010-10-05

    We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

  1. Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion.

    Science.gov (United States)

    Ravoo, B J; Weringa, W D; Engberts, J B

    2000-01-01

    Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se. Copyright 2000 Academic Press.

  2. Attachment of 99mTc to lipid vesicles containing the lipophilic chelate dipalmitoylphosphatidylethanolamine-DTPA

    International Nuclear Information System (INIS)

    Ahkong, Q.F.; Tilcock, C.

    1992-01-01

    The binding of 99m Tc to negatively-charged and neutral unilamellar lipid vesicles was investigated in the absence and presence of the ligand diethylenetriaminepentaacetic acid (DTPA) covalently attached to the headgroup of phosphatidylethanolamine at the surface of the membrane. Even in the absence of DTPA on the membrane surface, 99m Tc reduced by Sn bound to the membrane surface but rapidly dissociated from the vesicles in the presence of plasma in vitro. When DTPA was present on the membrane surface, dissociation of 99m Tc from the vesicle surface in plasma was much reduced. The dissociation of 99m Tc from the surface of negatively-charged vesicles was less than for neutral vesicles in the absence of ligand but was markedly greater than for vesicles containing the ligand DTPA, suggesting that the binding of 99m Tc to vesicles with surface-attached DTPA could not be explained solely on the basis of the negative charge provided by the DTPA. In vitro experiments using 14 C-labeled lipids as well as in vivo imaging studies indicated that dissociation of 99m Tc from the surface of the vesicle did not arise predominantly because of lipid exchange with plasma components or due to cleavage of Tc-DTPA from the vesicle surface. For vesicles with surface-attached DTPA, 99m Tc dissociation from the vesicle surface in plasma was further reduced by addition of the antioxidant ascorbate. (author)

  3. Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

    International Nuclear Information System (INIS)

    Stenovec, Matjaz; Kreft, Marko; Grilc, Sonja; Potokar, Maja; Kreft, Mateja Erdani; Pangrsic, Tina; Zorec, Robert

    2007-01-01

    Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca 2+ -dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca 2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca 2+ -triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles

  4. 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

    , an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations...... improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk...... of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved...

  5. Vesicle dynamics in a confined Poiseuille flow: from steady state to chaos.

    Science.gov (United States)

    Aouane, Othmane; Thiébaud, Marine; Benyoussef, Abdelilah; Wagner, Christian; Misbah, Chaouqi

    2014-09-01

    Red blood cells (RBCs) are the major component of blood, and the flow of blood is dictated by that of RBCs. We employ vesicles, which consist of closed bilayer membranes enclosing a fluid, as a model system to study the behavior of RBCs under a confined Poiseuille flow. We extensively explore two main parameters: (i) the degree of confinement of vesicles within the channel and (ii) the flow strength. Rich and complex dynamics for vesicles are revealed, ranging from steady-state shapes (in the form of parachute and slipper shapes) to chaotic dynamics of shape. Chaos occurs through a cascade of multiple periodic oscillations of the vesicle shape. We summarize our results in a phase diagram in the parameter plane (degree of confinement and flow strength). This finding highlights the level of complexity of a flowing vesicle in the small Reynolds number where the flow is laminar in the absence of vesicles and can be rendered turbulent due to elasticity of vesicles.

  6. Structure formation in binary mixtures of lipids and detergents: self-assembly and vesicle division.

    Science.gov (United States)

    Noguchi, Hiroshi

    2013-01-14

    Self-assembly dynamics in binary surfactant mixtures and structure changes of lipid vesicles induced by detergent solution are studied using coarse-grained molecular simulations. Disk-shaped micelles, the bicelles, are stabilized by detergents surrounding the rim of a bilayer disk of lipids. The self-assembled bicelles are considerably smaller than bicelles formed from vesicle rupture, and their size is determined by the concentrations of lipids and detergents and the interactions between the two species. The detergent-adsorption induces spontaneous curvature of the vesicle bilayer and results in vesicle division into two vesicles or vesicle rupture into worm-like micelles. The division occurs mainly via the inverse pathway of the modified stalk model. For large spontaneous curvature of the monolayers of the detergents, a pore is often opened, thereby leading to vesicle division or worm-like micelle formation.

  7. 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.

  8. Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

    Science.gov (United States)

    Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

    2016-04-01

    Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. The interactions between ionic surfactants and phosphatidylcholine vesicles: Conductometry

    Science.gov (United States)

    Tsao, Heng-Kwong; Tseng, Wen Liang

    2001-11-01

    The interaction between ionic surfactants and phosphatidylcholine vesicles, which are prepared without addition of buffer and salt, is investigated by conductivity measurements. On the basis of the vesicle acting as a trap of charge carriers, the bilayer/aqueous phase partition coefficient K and the surfactant/lipid molar ratio Re of nine surfactants are determined. The thermodynamic consistency is satisfied by the measured parameters. The effects of the alkyl chain length (C10-C16) and ionic head group are then studied. The inverse partition coefficient K-1 is linearly related to the critical micelle concentration. The solubilizing ability Reb is a consequence of the competition between the surfactant incorporation into the bilayer and the formation of micelles. Consequently, the K parameter rises whereas the Reb parameter declines as the chain length is increased. The influence due to addition of salt is also discussed.

  10. Thermal and active fluctuations of a compressible bilayer vesicle

    Science.gov (United States)

    Sachin Krishnan, T. V.; Yasuda, Kento; Okamoto, Ryuichi; Komura, Shigeyuki

    2018-05-01

    We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.

  11. 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.

  12. Importance of vesicle release stochasticity in neuro-spike communication.

    Science.gov (United States)

    Ramezani, Hamideh; Akan, Ozgur B

    2017-07-01

    Aim of this paper is proposing a stochastic model for vesicle release process, a part of neuro-spike communication. Hence, we study biological events occurring in this process and use microphysiological simulations to observe functionality of these events. Since the most important source of variability in vesicle release probability is opening of voltage dependent calcium channels (VDCCs) followed by influx of calcium ions through these channels, we propose a stochastic model for this event, while using a deterministic model for other variability sources. To capture the stochasticity of calcium influx to pre-synaptic neuron in our model, we study its statistics and find that it can be modeled by a distribution defined based on Normal and Logistic distributions.

  13. The Role of Extracellular Vesicles in Bone Metastasis

    Directory of Open Access Journals (Sweden)

    Michela Rossi

    2018-04-01

    Full Text Available Multiple types of cancer have the specific ability to home to the bone microenvironment and cause metastatic lesions. Despite being the focus of intense investigation, the molecular and cellular mechanisms that regulate the metastasis of disseminated tumor cells still remain largely unknown. Bone metastases severely impact quality of life since they are associated with pain, fractures, and bone marrow aplasia. In this review, we will summarize the recent discoveries on the role of extracellular vesicles (EV in the regulation of bone remodeling activity and bone metastasis occurrence. Indeed, it was shown that extracellular vesicles, including exosomes and microvesicles, released from tumor cells can modify the bone microenvironment, allowing the formation of osteolytic, osteosclerotic, and mixed mestastases. In turn, bone-derived EV can stimulate the proliferation of tumor cells. The inhibition of EV-mediated crosstalk between cancer and bone cells could represent a new therapeutic target for bone metastasis.

  14. Lubrication synergy: Mixture of hyaluronan and dipalmitoylphosphatidylcholine (DPPC) vesicles

    DEFF Research Database (Denmark)

    Raj, Akanksha; Wang, Min; Zander, Thomas

    2017-01-01

    consisting of non-homogeneous phospholipid bilayer with hyaluronan/DPPC aggregates on top. The presence of these aggregates generates a long-range repulsive surface force as two such surfaces are brought together. However, the aggregates are easily deformed, partly rearranged into multilayer structures......Phospholipids and hyaluronan have been implied to fulfil important roles in synovial joint lubrication. Since both components are present in synovial fluid, self-assembly structures formed by them should also be present. We demonstrate by small angle X-ray scattering that hyaluronan associates...... with the outer shell of dipalmitoylphophatidylcholine (DPPC) vesicles in bulk solution. Further, we follow adsorption to silica from mixed hyaluronan/DPPC vesicle solution by Quartz Crystal Microbalance with Dissipation measurements. Atomic Force Microscope imaging visualises the adsorbed layer structure...

  15. Pressure exerted by a vesicle on a surface

    International Nuclear Information System (INIS)

    Owczarek, A L; Prellberg, T

    2014-01-01

    Several recent works have considered the pressure exerted on a wall by a model polymer. We extend this consideration to vesicles attached to a wall, and hence include osmotic pressure. We do this by considering a two-dimensional directed model, namely that of area-weighted Dyck paths. Not surprisingly, the pressure exerted by the vesicle on the wall depends on the osmotic pressure inside, especially its sign. Here, we discuss the scaling of this pressure in the different regimes, paying particular attention to the crossover between positive and negative osmotic pressure. In our directed model, there exists an underlying Airy function scaling form, from which we extract the dependence of the bulk pressure on small osmotic pressures. (paper)

  16. Optimized microviscosimeter for detection and characterization of biological vesicles

    International Nuclear Information System (INIS)

    Gaiffe, O; Cretin, B; Boireau, W; Baudouy, J C; Vairac, P

    2008-01-01

    In this paper, we report on studies aimed at sensing the stiffness of biological membranes, in particular in the case of lipidic vesicles. To obtain pertinent results, we have developed and checked a specific sensor based on a vibrating sphere. The near-field acoustic wave generated by this vibrating sphere enables us to characterize biological particles which change the apparent viscosity and density of the surrounding fluid. The microsphere is well suited for very small volumes of liquid (typically about a few microlitres). The experimental results demonstrate the high sensitivity of the sensor to small variations of the composition of the aqueous media, particularly in the case of various populations of lipidic nanoparticles. Finally, this microviscosimeter demonstrates its ability to discriminate the population of vesicles on the basis of their global viscous properties

  17. A Phase of Liposomes with Entangled Tubular Vesicles

    Science.gov (United States)

    Chiruvolu, Shivkumar; Warriner, Heidi E.; Naranjo, Edward; Idziak, Stefan H. J.; Radler, Joachim O.; Plano, Robert J.; Zasadzinski, Joseph A.; Safinya, Cyrus R.

    1994-11-01

    An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled Ltv, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the Ltv phase coexists with the well-known L_4 phase of spherical vesicles and a bulk L_α phase. However, the defining characteristic of the Ltv phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure.

  18. The role of extracellular vesicles in phenotypic cancer transformation:

    OpenAIRE

    Kralj-Iglič, Veronika; Ogorevc, Eva; Veranič, Peter

    2013-01-01

    Background. Cancer has traditionally been considered as a disease resulting from gene mutations. New findings in biology are challenging gene-centered explanations of cancer progression and redirecting them to the non-genetic origins of tumorigenicity. It has become clear that intercellular communication plays a crucial role in cancer progression. Among the most intriguing ways of intercellular communication is that via extracellular vesicles (EVs). EVs are membrane structures released from v...

  19. Dimensional characterization of extracellular vesicles using atomic force microscopy

    International Nuclear Information System (INIS)

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

    2017-01-01

    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. (paper)

  20. Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

    Science.gov (United States)

    Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and

  1. Transfer of oleic acid between albumin and phospholipid vesicles

    International Nuclear Information System (INIS)

    Hamilton, J.A.; Cistola, D.P.

    1986-01-01

    The net transfer of oleic acid between egg phosphatidylcholine unilamellar vesicles and bovine serum albumin has been monitored by 13 C NMR spectroscopy and 90% isotopically substituted [1- 13 C]oleic acid. The carboxyl chemical shifts of oleic acid bound to albumin were different from those for oleic acid in phospholipid vesicles. Therefore, in mixtures of donor particles, the equilibrium distribution of oleic acid was determined from chemical shift and peak intensity data without separation of donor and acceptor particles. In a system containing equal masses of albumin and phospholipid and a stoichiometry of 4-5 mol of oleic acid per mol of albumin, the oleic acid distribution was pH dependent, with ≥80% of the oleic acid associated with albumin at pH 7.4; association was ≥90% at pH 8.0. Decreasing the pH below 7.4 markedly decreased the proportion of fatty acid bound to albumin. The distribution was reversible with pH and was independent of whether vesicles or albumin acted as a donor. These data suggest that pH may strongly influence the partitioning of fatty acid between cellular membranes and albumin. The 13 C NMR method is also advantageous because it provides information about the structural environments of oleic acid bound to albumin or phospholipid, the ionization state of oleic acid in each environment, and the structural integrity of the vesicles. In addition, minimum and maximum limits for the exchange rates of oleic acid among different environments were obtained from the NMR data

  2. Myeloid extracellular vesicles: messengers from the demented brain

    Directory of Open Access Journals (Sweden)

    Annamaria eNigro

    2016-01-01

    Full Text Available Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e. AD, PD, and ALS. Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases.

  3. Phase separation in artificial vesicles driven by light and curvature

    Science.gov (United States)

    Rinaldin, Melissa; Pomp, Wim; Schmidt, Thomas; Giomi, Luca; Kraft, Daniela; Physics of Life Processes Team; Soft; Bio Mechanics Collaboration; Self-Assembly in Soft Matter Systems Collaboration

    The role of phase-demixing in living cells, leading to the lipid-raft hypothesis, has been extensively studied. Lipid domains of higher lipid chain order are proposed to regulate protein spatial organization. Giant Unilamellar Vesicles provide an artificial model to study phase separation. So far temperature was used to initiate the process. Here we introduce a new methodology based on the induction of phase separation by light. To this aim, the composition of the lipid membrane is varied by photo-oxidation of lipids. The control of the process gained by using light allowed us to observe vesicle shape fluctuations during phase-demixing. The presence of fluctuations near the critical mixing point resembles features of a critical process. We quantitatively analyze these fluctuations using a 2d elastic model, from which we can estimate the material parameters such as bending rigidity and surface tension, demonstrating the non-equilibrium critical behaviour. Finally, I will describe recent attempts toward tuning the membrane composition by controlling the vesicle curvature.

  4. A Hierarchical Convolutional Neural Network for vesicle fusion event classification.

    Science.gov (United States)

    Li, Haohan; Mao, Yunxiang; Yin, Zhaozheng; Xu, Yingke

    2017-09-01

    Quantitative analysis of vesicle exocytosis and classification of different modes of vesicle fusion from the fluorescence microscopy are of primary importance for biomedical researches. In this paper, we propose a novel Hierarchical Convolutional Neural Network (HCNN) method to automatically identify vesicle fusion events in time-lapse Total Internal Reflection Fluorescence Microscopy (TIRFM) image sequences. Firstly, a detection and tracking method is developed to extract image patch sequences containing potential fusion events. Then, a Gaussian Mixture Model (GMM) is applied on each image patch of the patch sequence with outliers rejected for robust Gaussian fitting. By utilizing the high-level time-series intensity change features introduced by GMM and the visual appearance features embedded in some key moments of the fusion process, the proposed HCNN architecture is able to classify each candidate patch sequence into three classes: full fusion event, partial fusion event and non-fusion event. Finally, we validate the performance of our method on 9 challenging datasets that have been annotated by cell biologists, and our method achieves better performances when comparing with three previous methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Bacterial membrane vesicles, an overlooked environmental colloid: Biology, environmental perspectives and applications.

    Science.gov (United States)

    Toyofuku, Masanori; Tashiro, Yosuke; Hasegawa, Yusuke; Kurosawa, Masaharu; Nomura, Nobuhiko

    2015-12-01

    Phospholipid vesicles play important roles in biological systems. Bacteria are one of the most abundant organisms on Earth, and bacterial membrane vesicles (MVs) were first observed 50 years ago. Many bacteria release MVs to the environment that mainly consist of the cell membrane and typically range from 20 to 400 nm in size. Bacterial MVs are involved in several biological functions, such as delivery of cargo, virulence and gene transfer. MVs can be isolated from laboratory culture and directly from the environment, indicating their high abundance in and impact on ecosystems. Many colloidal particles in the environment ranging in size from 1 nm to 1 μm have been reported but not characterized at the molecular level, and MVs remain to be explored. Hence, MVs can be considered terra incognita in environmental colloid research. Although MV biogenesis and biological roles are yet to be fully understood, the accumulation of knowledge has opened new avenues for their applications. Via genetic engineering, the MV yield can be greatly increased, and the components of MVs can be tailored. Recent studies have demonstrated that MVs have promising potential for applications such as drug delivery systems and nanobiocatalysts. For instance, MV vaccines have been extensively studied and have already been approved in Europe. Recent MV studies have evoked great interest in the fields of biology and biotechnology, but fundamental questions, such as their transport in the environment or physicochemical features of MVs, remain to be addressed. In this review, we present the current understanding of bacterial MVs and environmental perspectives and further introduce their applications. Copyright © 2015. Published by Elsevier B.V.

  6. Cellular therapy without cells: extracellular vesicles promote activation of stem cells after irradiation

    International Nuclear Information System (INIS)

    Lange, C.

    2016-01-01

    Mesenchymal stromal cells from the bone marrow (MSC) have been shown to be effective in several cell therapeutic treatments. However, MSC accumulate in lungs after i.v. injection. How do MSC transfer their potential to organs with therapeutic need? We show that released extracellular vesicles (EV) might be playing an active role in this transfer. EV were isolated from MSC supernatant and characterized with flow cytometry, proteomics and next generation sequencing. Our data showed the transfer of RNAs, clustering into several protective gene groups. Besides, we repeatedly detected genomic DNA on vesicles. Using a plant - derived detector gene we showed horizontal DNA transfer via EV. Furthermore, we showed that EV were able to salvage stem/progenitor cells in vitro from radiation suppression. Three selected proteins from proteomics data were examined for stem cell protection after irradiation. EV derived from down-regulated producer MSC showed a substantial loss of protection in irradiated stem cells supporting their relevance for stem cell protection. Finally, we showed that EV after i.v. injection into lethally irradiated animals colocalize within 2-4 hours with hematopoietic stem cells in the bone marrow giving hint to direct protection of stem cells by EV. In conclusion, EV derived from bone marrow MSC were able to transfer several cargo compounds leading potentially to change of the genetic properties. Importantly, EV protect irradiated hematopoietic stem cells, stimulate their recovery and proliferation and rescue lethally irradiated animals long-term. Thus, EV might be an alternative for future cell therapeutic treatment particularly in radiation-based events. (author)

  7. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination.

    Science.gov (United States)

    Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

    2016-01-01

    Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with

  8. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  9. Use of membrane vesicles as a simplified system for studying auxin transport of auxin: Progress report

    International Nuclear Information System (INIS)

    Goldsmith, M.H.M.

    1986-01-01

    Indoleacetic acid (IAA), the auxin regulating growth, is transported polarly in plants. IAA stimulates a rapid increase in the rate of electrogenic proton secretion by the plasma membrane. This not only increases the magnitude of the pH and electrical gradients providing the driving force for polar auxin transport and uptake of sugars, amino acids and inorganic ions, but, by acidifying the cell wall, also leads to growth. We find that auxin uptake by membrane vesicles isolated from actively growing plant tissues exhibits some of the same properties as by cells: the accumulation depends on the pH gradient, is saturable and specific for auxin, and enhanced by herbicides that inhibit polar auxin transport. We are using accumulation of a radioactive weak acid to quantify the pH gradient and distribution of fluorescent cyanine dyes to monitor the membrane potential. The magnitude of IAA accumulation exceeds that predicted from the pH gradient, and in the absence of a pH gradient, a membrane potential fails to support any auxin accumulation, leading to the conclusion that the transmembrane potential is not a significant driving force for auxin accumulation in this system. Since increasing the external ionic strength decreases saturable auxin accumulation, we are investigating how modifying the surface potential of the vesicles affects the interaction of the amphipathic IAA molecules with the membranes and whether protein modifying reagents affect the saturability and stimulation by NPA. These studies should provide information on the location and function of the auxin binding site and may enable us to identify the solubilized protein. 5 refs

  10. Optimized exosome isolation protocol for cell culture supernatant and human plasma

    Directory of Open Access Journals (Sweden)

    Richard J. Lobb

    2015-07-01

    Full Text Available Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a

  11. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.

    Science.gov (United States)

    Wang, Juan; Silva, Malan; Haas, Leonard A; Morsci, Natalia S; Nguyen, Ken C Q; Hall, David H; Barr, Maureen M

    2014-03-03

    Cells release extracellular vesicles (ECVs) that play important roles in intercellular communication and may mediate a broad range of physiological and pathological processes. Many fundamental aspects of ECV biogenesis and signaling have yet to be determined, with ECV detection being a challenge and obstacle due to the small size (100 nm) of the ECVs. We developed an in vivo system to visualize the dynamic release of GFP-labeled ECVs. We show here that specific Caenorhabdidits elegans ciliated sensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are also abundant in the lumen surrounding the cilium. Electron tomography and genetic analysis indicate that ECV biogenesis occurs via budding from the plasma membrane at the ciliary base and not via fusion of multivesicular bodies. Intraflagellar transport and kinesin-3 KLP-6 are required for environmental release of PKD-2::GFP-containing ECVs. ECVs isolated from wild-type animals induce male tail-chasing behavior, while ECVs isolated from klp-6 animals and lacking PKD-2::GFP do not. We conclude that environmentally released ECVs play a role in animal communication and mating-related behaviors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  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. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    Science.gov (United States)

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  14. Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform

    Directory of Open Access Journals (Sweden)

    M. Fátima Barroso

    2016-05-01

    Full Text Available In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC and 1-undecanethiol (SH. After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH–DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures.

  15. Porphyromonas gingivalis Outer Membrane Vesicles Mediate Coaggregation and Piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum

    Directory of Open Access Journals (Sweden)

    Daniel Grenier

    2013-01-01

    Full Text Available Porphyromonas gingivalis sheds outer membrane vesicles that contain several virulence factors, including adhesins. In this study, we investigated the ability of P. gingivalis outer membrane vesicles to mediate the coaggregation and piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum. Marked coaggregation between T. denticola and L. saburreum occurred in the presence of P. gingivalis outer membrane vesicles. Sucrose was an effective chemoattractant for the motile species T. denticola. The addition of outer membrane vesicles to a mixture of T. denticola and L. saburreum significantly increased the number of nonmotile bacteria that migrated into a sucrose-filled capillary tube immersed in the bacterial mixture. Under optimal conditions, the number of nonmotile L. saburreum in the capillary tube increased approximately 5-fold, whereas no increase occurred when boiled vesicles were used. This study showed that P. gingivalis outer membrane vesicles mediate coaggregation between T. denticola and L. saburreum and that nonmotile bacteria can be translocated by piggybacking on spirochetes.

  16. Replication of simulated prebiotic amphiphile vesicles controlled by experimental lipid physicochemical properties

    International Nuclear Information System (INIS)

    Armstrong, Don L; Zidovetzki, Raphael; Markovitch, Omer; Lancet, Doron

    2011-01-01

    We present a new embodiment of the graded autocatalysis replication domain (GARD) for the growth, replication and evolution of lipid vesicles based on a semi-empirical foundation using experimentally measured kinetic values of selected extant lipid species. Extensive simulations using this formalism elucidated the details of the dependence of the replication and properties of the vesicles on the physicochemical properties and concentrations of the lipids, both in the environment and in the vesicle. As expected, the overall concentration and number of amphiphilic components strongly affect average replication time. Furthermore, variations in acyl chain length and unsaturation of vesicles also influence replication rate, as do the relative concentrations of individual lipid types. Understanding of the dependence of replication rates on physicochemical parameters opens a new direction in the study of prebiotic vesicles and lays the groundwork for future studies involving the competition between lipid vesicles for available amphiphilic monomers

  17. 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.

  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

    The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection...... 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...... dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results...

  19. Small round blue cell tumor of seminal vesicle in a young patient

    Directory of Open Access Journals (Sweden)

    Adriano A. De Paula

    2006-10-01

    Full Text Available Seminal vesicle tumor is a rare disease with unclear origin. Generally, it is presented as a pelvic mass that can be detected by sonography and digital rectal exam. The authors report a 25-year-old patient with a pelvic mass which the magnetic resonance and surgical specimen reveal a seminal vesicle tumor. Immunohistochemical findings favored a primitive neuroectodermal tumor of the seminal vesicle. Herein, the treatment, histological and histochemical findings of this entity are discussed.

  20. Synthesis of Copper nanoparticles through vesicle template using gamma irradiation

    International Nuclear Information System (INIS)

    Noor Ezzah Rahimah Ahmad Samsuri

    2012-01-01

    Nano technology has gained attention for its application in life. This study was conducted to produce copper (Cu) nanoparticles using gamma ray irradiation through template vesicles. Cu nanoparticle has a variety of applications such as capacitor materials, catalyst, conductive coating, high thermal conductivity materials as well as lubricant additives. this study used gamma radiation compared to other methods because the use of gamma rays in producing nanoparticle is safer and environmental friendly. The purpose of this study was to see the effects of radiation on the formation of Cu nanoparticles. The radiation dose used was 80 kGy and 100 kGy. The vesicles were formed by mixing water, sodium n-lauroyl sarcosinat hydrated, 1-decanol and polyethylene glycol with certain ratio (85 %: 5 %: 7 %: 3 %). Analysis from the transmission electron microscopy (TEM) showed the production of multilammelar vesicles in size between 30 nm-80 nm. The formation of nanoparticles was analyzed using UV-Vis absorption spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy (TEM). Analysis of UV-Vis absorption spectroscopy showed no resonance peak around 600 nm. XRD analysis confirmed the presence of Cu, Cu 2 O and CuO. Analysis and characterisation using transmission electron microscopy (TEM) also confirmed that nanoparticles were produced with different sizes according to the radiation dose. At the radiation dose of 80 kGy, nanoparticles size is found vary between 30 nm to 90 nm. While at the radiation dose of 100 kGy, nanoparticles size is found vary between 3 nm to 7 nm. From this study it can be concluded that higher radiation dse will produce smaller nanoparticles. (author)

  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. Selective Metal-Ion-Mediated Vesicle Adhesion Based on Dynamic Self-Organization of a Pyrene-Appended Glutamic Acid.

    Science.gov (United States)

    Xing, Pengyao; Wang, Yajie; Yang, Minmin; Zhang, Yimeng; Wang, Bo; Hao, Aiyou

    2016-07-13

    Vesicles with dynamic membranes provide an ideal model system for investigating biological membrane activities, whereby vesicle aggregation behaviors including adhesion, fusion, fission, and membrane contraction/extension have attracted much attention. In this work we utilize an aromatic amino acid (pyrene-appended glutamic acid, PGlu) to prepare nanovesicles that aggregate to form vesicle clusters selectively induced by Fe(3+) or Cu(2+), and the vesicles transform into irregular nano-objects when interacting with Al(3+). Vesicle clusters have better stability than pristine vesicles, which hinders the spontaneous morphological transformation from vesicles into lamellar nanosheets with long incubation period. The difference between complexation of Fe(3+) and Al(3+) with vesicles was studied by various techniques. On the basis of metal ion-vesicle interactions, this self-assembled nanovesicle system also behaves as an effective fluorescent sensor for Fe(3+) and Al(3+), which cause fluorescence quenching and enhanced excimer emission, respectively.

  3. Chromatic response of polydiacetylene vesicle induced by the permeation of methotrexate.

    Science.gov (United States)

    Shin, Min Jae; Kim, Ye Jin; Kim, Jong-Duk

    2015-07-07

    The noble vesicular system of polydiacetylene showed a red shift using two types of detecting systems. One of the systems involves the absorption of target materials from the outer side of the vesicle, and the other system involves the permeation through the vesicular layers from within the vesicle. The chromatic mixed vesicles of N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA) and dimethyldioctadecylammonium chloride (DODAC) were fabricated by sonication, followed by polymerization by UV irradiation. The stability of monomeric vesicles was observed to increase with the polymerization of the vesicles. Methotrexate was used as a target material. The polymerized mixed vesicles having a blue color were exposed to a concentration gradient of methotrexate, and a red shift was observed indicating the adsorption of methotrexate on the polydiacetylene bilayer. In order to check the chromatic change by the permeation of methotrexate, we separated the vesicle portion, which contained methotrexate inside the vesicle, and checked chromatic change during the permeation of methotrexate through the vesicle. The red shift apparently indicates the disturbance in the bilayer induced by the permeation of methotrexate. The maximum contrast of color appeared at the equal molar ratio of AEPCDA and DODAC, indicating that the formation of flexible and deformable vesicular layers is important for red shift. Therefore, it is hypothesized that the system can be applicable for the chromatic detection of the permeation of methotrexate through the polydiacetylene layer.

  4. Dynamics of coarsening in multicomponent lipid vesicles with non-uniform mechanical properties

    Science.gov (United States)

    Funkhouser, Chloe M.; Solis, Francisco J.; Thornton, K.

    2014-04-01

    Multicomponent lipid vesicles are commonly used as a model system for the complex plasma membrane. One phenomenon that is studied using such model systems is phase separation. Vesicles composed of simple lipid mixtures can phase-separate into liquid-ordered and liquid-disordered phases, and since these phases can have different mechanical properties, this separation can lead to changes in the shape of the vesicle. In this work, we investigate the dynamics of phase separation in multicomponent lipid vesicles, using a model that couples composition to mechanical properties such as bending rigidity and spontaneous curvature. The model allows the vesicle surface to deform while conserving surface area and composition. For vesicles initialized as spheres, we study the effects of phase fraction and spontaneous curvature. We additionally initialize two systems with elongated, spheroidal shapes. Dynamic behavior is contrasted in systems where only one phase has a spontaneous curvature similar to the overall vesicle surface curvature and systems where the spontaneous curvatures of both phases are similar to the overall curvature. The bending energy contribution is typically found to slow the dynamics by stabilizing configurations with multiple domains. Such multiple-domain configurations are found more often in vesicles with spheroidal shapes than in nearly spherical vesicles.

  5. 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.

  6. Tension-induced vesicle fusion: pathways and pore dynamics

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2008-01-01

    and eventually opens a pore to complete the fusion process. In pathway II, at higher tension, a stalk is formed during the fusion process that is then transformed by transmembrane pore formation into a fusion pore. Whereas the latter pathway II resembles stalk pathways as observed in other simulation studies......, fusion pathway I, which does not involve any stalk formation, has not been described previously to the best of our knowledge. A statistical analysis of the various processes shows that fusion is the dominant pathway for releasing the tension of the vesicles. The functional dependence of the observed...

  7. Extracellular Vesicles in Heart Disease: Excitement for the Future?

    Directory of Open Access Journals (Sweden)

    Kirsty M. Danielson

    2014-01-01

    Full Text Available Extracellular vesicles (EV, including exosomes, microvesicles and apoptotic bodies, are released from numerous cell types and are involved in intercellular communication, physiological functions and the pathology of disease. They have been shown to carry and transfer a wide range of cargo including proteins, lipids and nucleic acids. The role of EVs in cardiac physiology and heart disease is an emerging field that has produced intriguing findings in recent years. This review will outline what is currently known about EVs in the cardiovascular system, including cellular origins, functional roles and utility as biomarkers and potential therapeutics.

  8. Mechanism of ochratoxin A transport in kidney

    International Nuclear Information System (INIS)

    Sokol, P.P.; Ripich, G.; Holohan, P.D.; Ross, C.R.

    1988-01-01

    The effect of the fungal metabolite (mycotoxin) Ochratoxin A (OTA) on the transport of p-amino[ 3 H]hippurate (PAH), a prototypic organic anion, was examined in renal brush border (BBMV) and basolateral membrane vesicles (BLMV). OTA was as effective an inhibitor of PAH uptake in both membranes as probenecid. The dose response curves for OTA in BBMV and BLMV gave IC50 values of 20 +/- 6 and 32 +/- 7 microM, respectively. The effect was specific since the transport of the organic cation N1-methylnicotinamide was not affected. The phenomenon of counterflow was studied to establish that OTA is translocated. OTA produced trans stimulation of PAH transport in both BBMV and BLMV, demonstrating that OTA is transported across both these membranes. The data suggest that OTA interacts with the PAH transport system in both BBMV and BLMV. We conclude that OTA transport in the kidney is mediated via the renal organic anion transport system

  9. Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

    Science.gov (United States)

    Lerner, Natalie; Avissar, Sofia; Beit-Yannai, Elie

    2017-01-01

    Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of

  10. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

    International Nuclear Information System (INIS)

    Rajendran, V.M.; Kashgarian, M.; Binder, H.J.

    1989-01-01

    Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats

  11. Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

    Science.gov (United States)

    Semple, S C; Klimuk, S K; Harasym, T O; Dos Santos, N; Ansell, S M; Wong, K F; Maurer, N; Stark, H; Cullis, P R; Hope, M J; Scherrer, P

    2001-02-09

    Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

  12. Isolation and characterization of plasma membranes from guinea pig ileum

    International Nuclear Information System (INIS)

    Anon.

    1986-01-01

    A plasma membrane fraction from guinea pig ileum has been isolated by extraction of a crude microsomal fraction with a low ionic strength buffer containing ATP and Ca 2+ . The extracted microsomes were subjected to sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membranes were substantially free from contamination with contractile proteins, mitochondria and sarco-plasmic reticulum. The plasma membrane vesicles were enriched 30-to-40-fold in Na + -K + -ATPase and 5'-nucleotidase activities. The plasma membrane vesicles accumulated Ca 2+ in the presence of ATP. The addition of Ca 2+ ionophore A23187 to vesicles loaded with Ca 2+ in the presence of ATP removed Ca 2+ completely from the vesicles in one minute. The Km values for the Ca 2+ -dependent phosphorylated intermediates of Ca 2+ -Mg 2+ -ATPase and Ca 2+ uptake were approximately 0.8 μM indicating that the phosphorylated intermediates represent phosphorylation of Ca 2+ pump ATPase. The 3 H-nitrendipine binding to plasma membranes was characterized by high affinity with Kd of 185 pM and B/sub max/ 1280 fmol/mg protein. The plasma membrane vesicles prepared by these procedures can prove useful for the study of ion transport

  13. Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

    Science.gov (United States)

    Owen, P; Salton, M R

    1975-10-06

    The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.

  14. Bicarbonate sulfate exchange in canalicular rat liver plasma membrane vesicles

    International Nuclear Information System (INIS)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I.

    1987-01-01

    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 [ 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'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC 50 , ∼40 μM). 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

  15. The computational route from bilayer membranes to vesicle fusion

    International Nuclear Information System (INIS)

    Shillcock, Julian C; Lipowsky, Reinhard

    2006-01-01

    Biological membranes are examples of 'smart' materials whose properties and behaviour emerge from the propagation across many scales of the molecular characteristics of their constituents. Artificial smart materials, such as drug delivery vehicles and biosensors, often rely on modifying naturally occurring soft matter, such as polymers and lipid vesicles, so that they possess useful behaviour. However, the complexity of natural membranes, both in their static properties, exemplified in their phase behaviour, and in their dynamic properties, as in the kinetics of their formation and interactions, hinders their rational modification. Mesoscopic simulations, such as dissipative particle dynamics (DPD), allow in silico experiments to be easily and cheaply performed on complex, soft materials requiring as input only the molecular structure of the constituents at a coarse-grained level. They can therefore act as a guide to experimenters prior to performing costly assays. Additionally, mesoscopic simulations provide the only currently feasible window on the length- and timescales relevant to important biophysical processes such as vesicle fusion. We review here the development of computational models of bilayer membranes, and in particular the use of mesoscopic simulations to follow the molecular rearrangements that occur during membrane fusion

  16. Intracellular vesicle acidification promotes maturation of infectious poliovirus particles.

    Directory of Open Access Journals (Sweden)

    Alexsia L Richards

    Full Text Available The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time bona fide autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.

  17. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  18. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  19. Intraluminal proteome and peptidome of human urinary extracellular vesicles.

    Science.gov (United States)

    Liu, Xinyu; Chinello, Clizia; Musante, Luca; Cazzaniga, Marta; Tataruch, Dorota; Calzaferri, Giulio; James Smith, Andrew; De Sio, Gabriele; Magni, Fulvio; Zou, Hequn; Holthofer, Harry

    2015-06-01

    Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels. UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS. A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content. Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Vesicle computers: Approximating a Voronoi diagram using Voronoi automata

    International Nuclear Information System (INIS)

    Adamatzky, Andrew; De Lacy Costello, Ben; Holley, Julian; Gorecki, Jerzy; Bull, Larry

    2011-01-01

    Highlights: → We model irregular arrangements of vesicles filled with chemical systems. → We examine influence of precipitation threshold on the system's computational potential. → We demonstrate computation of Voronoi diagram and skeleton. - Abstract: Irregular arrangements of vesicles filled with excitable and precipitating chemical systems are imitated by Voronoi automata - finite-state machines defined on a planar Voronoi diagram. Every Voronoi cell takes four states: resting, excited, refractory and precipitate. A resting cell excites if it has at least one neighbour in an excited state. The cell precipitates if the ratio of excited cells in its neighbourhood versus the number of neighbours exceeds a certain threshold. To approximate a Voronoi diagram on Voronoi automata we project a planar set onto the automaton lattice, thus cells corresponding to data-points are excited. Excitation waves propagate across the Voronoi automaton, interact with each other and form precipitate at the points of interaction. The configuration of the precipitate represents the edges of an approximated Voronoi diagram. We discover the relationship between the quality of the Voronoi diagram approximation and the precipitation threshold, and demonstrate the feasibility of our model in approximating Voronoi diagrams of arbitrary-shaped objects and in constructing a skeleton of a planar shape.

  1. Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

    Directory of Open Access Journals (Sweden)

    Hina Kalra

    Full Text Available Extracellular vesicles (EVs are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

  2. 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.

  3. Exocytosis from chromaffin cells: hydrostatic pressure slows vesicle fusion

    Science.gov (United States)

    Stühmer, Walter

    2015-01-01

    Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å3 for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes. PMID:26009771

  4. Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1.

    Science.gov (United States)

    Gracheva, Elena O; Maryon, Ed B; Berthelot-Grosjean, Martine; Richmond, Janet E

    2010-01-01

    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 25 nm 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.

  5. Composition Effect of the Outer Layer on the Vesicle Fusion Catalyzed by Phospholipase D

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jin Won [Seoul National University, Seoul (Korea, Republic of)

    2014-09-15

    Phospholipase D (PLD) catalyzed the generation of phosphatidic acid (PA) from phosphatidylcholine (PC) at the outer layer of the vesicles prepared through layer by layer via a double emulsion technique. The generation induced a curvature change in the vesicles, which eventually led them to fuse each other. The ratio of two-fattyacid-tail ethanolamine (PE) to one-fatty-acid-tail ethanolamine (PE) was found to acquire the condition where the mixed-phospholipid vesicles were stable identically with pure two-fatty-acid-tail PC. The effect of the outer-layer mixture on the PLD-induced vesicle fusion was investigated using the fluorescence intensity change. 8-Aminonaph- thalene-1,3,6-trisulfonic acid disodium salt (ANTS) and p-Xylene-bis(N-pyridinium bromide) (DPX) were encapsulated in the vesicles, respectively, for the quantification of the fusion. The fluorescence scale was calibrated with the fluorescence of a 1/1 mixture of ANTS and DPX vesicles in NaCl buffer taken as 100% fluorescence (0% fusion) and the vesicles containing both ANTS and DPX as 0% fluorescence (100% fusion), considering the leakage into the medium studied directly in a separate experiment using vesicles containing both ANTS and DPX. The fusion data for each composition were acquired with the subtraction of the leakage from the quenching. From the monitoring, the vesicle fusion caused by the PLD reaction seems dominantly to occur rather than the vesicle lysis, because the composition effect on the fusion was observed identically with that on the change in the vesicle structure. Furthermore, the diameter measurements also support the fusion dominancy.

  6. Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

    Science.gov (United States)

    Flavin, William P; Bousset, Luc; Green, Zachary C; Chu, Yaping; Skarpathiotis, Stratos; Chaney, Michael J; Kordower, Jeffrey H; Melki, Ronald; Campbell, Edward M

    2017-10-01

    Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

  7. 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.

  8. 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

  9. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Jan Lötvall

    2014-12-01

    Full Text Available Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs, which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

  10. Frequency-dependent electrodeformation of giant phospholipid vesicles in AC electric field

    Science.gov (United States)

    2010-01-01

    A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modeled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kilohertz range and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied alternating current electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys J 95:L19–L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed. PMID:21886342

  11. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

    Science.gov (United States)

    Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

    2018-01-01

    Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

  12. G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

    Science.gov (United States)

    Photowala, Huzefa; Blackmer, Trillium; Schwartz, Eric; Hamm, Heidi E; Alford, Simon

    2006-03-14

    Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

  13. The early postnatal pattern of vesicle formation in different regions of the porcien small intestine

    DEFF Research Database (Denmark)

    Elbrønd, Vibeke Sødring; Weström, B.R.

    2007-01-01

    applied to visualize cytoplasmatic subcellular components such as fat (Oil red O) and carbohydrates (PAS). Appearance and morphology of the epithelial vesicles were compared. In the proximal region several small supranuclear and a single large subnuclear electron dense, eosinophilic and PAS+ vesicle were...

  14. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance

    DEFF Research Database (Denmark)

    Holst, Birgitte; Madsen, Kenneth L; Jansen, Anna M

    2013-01-01

    by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate...

  15. Magnetic resonance of seminal vesicles: a noninvasive study of seminal way

    International Nuclear Information System (INIS)

    Ocantos, J.A.; Rey Valzacchi, G.; Sinclair, M.E.; Loor Guadamud, G.

    2010-01-01

    The magnetic resonance without endorectal coil is an excellent diagnostic tool for studying the entire route of seminal non-invasive way in a single step diagnosis. We call magnetic resonance of seminal vesicles, but includes both the study of the seminal vesicles as the channels of the seminal way. [es

  16. Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission.

    Science.gov (United States)

    Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A

    2013-06-15

    Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction 'buds' suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior.

  17. Molecular dynamics simulation of the formation, structure, and dynamics of small phospholipid vesicles

    NARCIS (Netherlands)

    Marrink, SJ; Mark, AE

    2003-01-01

    Here, we use coarse grained molecular dynamics (MD) simulations to study the spontaneous aggregation of dipalmitoylphosphatidylcholine (DPPC) lipids into small unilamellar vesicles. We show that the aggregation process occurs on a nanosecond time scale, with bicelles and cuplike vesicles formed at

  18. Polymerization of styrene in DODAB vesicles : a small-angle neutron scattering study

    NARCIS (Netherlands)

    Jung, M.; Robinson, B.H.; Steytler, D.C.; German, A.L.; Heenan, R.K.

    2002-01-01

    The polymerization of styrene, located in the bilayer of dioctadecyldimethylammonium bromide (DODAB) vesicles, gives rise to phase separation between the growing polymer and the bilayer. The result is a small (20-30 nm) bead of polymer located in the bilayer of each vesicle giving them a

  19. Adrenomedullin increases the short-circuit current in the mouse seminal vesicle: actions on chloride secretion.

    Science.gov (United States)

    Liao, S B; Cheung, K H; O, W S; Tang, Fai

    2014-08-01

    Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility. © 2014 by the Society for the Study of Reproduction, Inc.

  20. Dynamics of fatty acid vesicles in response to pH stimuli

    DEFF Research Database (Denmark)

    Ikari, Keita; Sakuma, Yuka; Jimbo, Takehiro

    2015-01-01

    We investigate the dynamics of decanoic acid/decanoate (DA) vesicles in response to pH stimuli. Two types of dynamic processes induced by the micro injection of NaOH solutions are sequentially observed: deformations and topological transitions. In the deformation stage, DA vesicles show a series...

  1. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

    DEFF Research Database (Denmark)

    Shupliakov, Oleg; Bloom, Ona; Gustafsson, Jenny S

    2002-01-01

    Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin ...

  2. Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Hadorn, M.; Boenzli, E.; Sørensen, Kristian T.

    2013-01-01

    The technological aspects of artificial vesicles as prominent cell mimics are evolving toward higher-order assemblies of functional vesicles with tissuelike architectures. Here, we demonstrate the spatially controlled DNA-directed bottom-up synthesis of complex microassemblies and macroassemblies...

  3. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  4. Origins of microstructural transformations in charged vesicle suspensions: the crowding hypothesis.

    Science.gov (United States)

    Seth, Mansi; Ramachandran, Arun; Murch, Bruce P; Leal, L Gary

    2014-09-02

    It is observed that charged unilamellar vesicles in a suspension can spontaneously deflate and subsequently transition to form bilamellar vesicles, even in the absence of externally applied triggers such as salt or temperature gradients. We provide strong evidence that the driving force for this deflation-induced transition is the repulsive electrostatic pressure between charged vesicles in concentrated suspensions, above a critical effective volume fraction. We use volume fraction measurements and cryogenic transmission electron microscopy imaging to quantitatively follow both the macroscopic and microstructural time-evolution of cationic diC18:1 DEEDMAC vesicle suspensions at different surfactant and salt concentrations. A simple model is developed to estimate the extent of deflation of unilamellar vesicles caused by electrostatic interactions with neighboring vesicles. It is determined that when the effective volume fraction of the suspension exceeds a critical value, charged vesicles in a suspension can experience "crowding" due to overlap of their electrical double layers, which can result in deflation and subsequent microstructural transformations to reduce the effective volume fraction of the suspension. Ordinarily in polydisperse colloidal suspensions, particles interacting via a repulsive potential transform into a glassy state above a critical volume fraction. The behavior of charged vesicle suspensions reported in this paper thus represents a new mechanism for the relaxation of repulsive interactions in crowded situations.

  5. Yarrowia lipolytica vesicle-mediated protein transport pathways

    Directory of Open Access Journals (Sweden)

    Beckerich Jean-Marie

    2007-11-01

    Full Text Available Abstract Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii. These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular

  6. v-SNAREs control exocytosis of vesicles from priming to fusion.

    Science.gov (United States)

    Borisovska, Maria; Zhao, Ying; Tsytsyura, Yaroslav; Glyvuk, Nataliya; Takamori, Shigeo; Matti, Ulf; Rettig, Jens; Südhof, Thomas; Bruns, Dieter

    2005-06-15

    SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

  7. Folding Up of Gold Nanoparticle Strings into Plasmonic Vesicles for Enhanced Photoacoustic Imaging

    KAUST Repository

    Liu, Yijing

    2015-11-11

    The stepwise self-assembly of hollow plasmonic vesicles with vesicular membranes containing strings of gold nanoparticles (NPs) is reported. The formation of chain vesicles can be controlled by tuning the density of the polymer ligands on the surface of the gold NPs. The strong absorption of the chain vesicles in the near-infrared (NIR) region leads to a much higher efficiency in photoacoustic (PA) imaging than for non-chain vesicles. The chain vesicles were further employed for the encapsulation of drugs and the NIR light triggered release of payloads. This work not only offers a new platform for controlling the hierarchical self-assembly of NPs, but also demonstrates that the physical properties of the materials can be tailored by controlling the spatial arrangement of NPs within assemblies to achieve a better performance in biomedical applications.

  8. Two types of mineral-related matrix vesicles in the bone mineralization of zebrafish

    International Nuclear Information System (INIS)

    Yang, L; Zhang, Y; Cui, F Z

    2007-01-01

    Two types of mineral-related matrix vesicle, multivesicular body (MVB) and monovesicle, were detected in the skeletal bone of zebrafish. Transmission electron microscopy and energy dispersive spectroscopy (EDS) analyses of the vesicular inclusions reveal that both types of vesicles contain calcium and phosphorus, suggesting that these vesicles may be involved in mineral ion delivery for the bone mineralization of zebrafish. However, their size and substructure are quite different. Monovesicles, whose diameter ranges from 100 nm to 550 nm, are similar to the previously reported normal matrix vesicles, while MVBs have a larger size of 700-1000 nm in nominal diameter and possess a substructure that is composed of smaller vesicles with their average size around 100 nm. The presence of mineral-related MVBs, which is first identified in zebrafish bone, indicates that the mineralization-associated transportation process of mineral ions is more complicated than is ordinarily imagined

  9. Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

    Science.gov (United States)

    Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

    2014-01-01

    Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

  10. Investigation of vesicle-capsular plague antigen complex formation by elastic laser radiation scattering

    Science.gov (United States)

    Guseva, N. P.; Maximova, Irina S.; Romanov, Sergey V.; Shubochkin, L. P.; Tatarintsev, Sergey N.

    1991-05-01

    Recently a great deal of attention has been given to the investigation artificial lipid liposomes, due to their application as "containers" for directed transport of biologically active compounds into particular cells, organs and tissues for prophylaxis and therapy of infectious diseases. The use of traditional methods of liposome investigation, such as sedimentation, electrophoresis and chromatography is impeded by low liposome resistivity to different deformations. In conjunction with this, optical methods of laser light scattering are promising as they allow nondisturbing, precise and quick investigations. This paper describes the investigation of vesicle systems prepared from egg lecithin of Serva Corporation and their complexes with the capsular antigen of the plague microbe. The capsular antigen Fl was obtained from EV plague microbe grown at 37° C on Huttinger agar. Fl was isolated by gel-filtration on ASA-22 followed by freeze drying of the preparation. Angular dependences of polarized radiation scattering were measured for several liposome suspension samples in a saline solution before and after the interaction with the plague microbe capsular antigen. The aim of the investigation was to analyze the nature of mutual antigen arrangement in a liposome and to develop methods for measuring its inclusion percentage.

  11. Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes

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    Maja Mustapic

    2017-05-01

    Full Text Available Our team has been a pioneer in harvesting extracellular vesicles (EVs enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimer's and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum, or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EVs enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner.

  12. Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy

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    Johanna L. Höög

    2015-11-01

    Full Text Available Human ejaculates contain extracellular vesicles (EVs, that to a large extent are considered to originate from the prostate gland, and are often denominated “prostasomes.” These EVs are important for human fertility, for example by promoting sperm motility and by inducing immune tolerance of the female immune system to the spermatozoa. So far, the EVs present in human ejaculate have not been studied in their native state, inside the seminal fluid without prior purification and isolation procedures. Using cryo-electron microscopy and tomography, we performed a comprehensive inventory of human ejaculate EVs. The sample was neither centrifuged, fixed, filtered or sectioned, nor were heavy metals added. Approximately 1,500 extracellular structures were imaged and categorized. The extracellular environment of human ejaculate was found to be diverse, with 5 major subcategories of EVs and 6 subcategories of extracellular membrane compartments, including lamellar bodies. Furthermore, 3 morphological features, including electron density, double membrane bilayers and coated surface, are described in all subcategories. This study reveals that the extracellular environment in human ejaculate is multifaceted. Several novel morphological EV subcategories are identified and clues to their cellular origin may be found in their morphology. This inventory is therefore important for developing future experimental approaches, and to interpret previously published data to understand the role of EVs for human male fertility.

  13. Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients

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    Samuel I. Anyanwu

    2018-01-01

    Full Text Available Background. Extracellular vesicles (EVs are membrane bound, secreted by cells, and detected in bodily fluids, including urine, and contain proteins, RNA, and DNA. Our goal was to identify HIV and human proteins (HPs in urinary EVs from HIV+ patients and compare them to HIV− samples. Methods. Urine samples were collected from HIV+ (n=35 and HIV− (n=12 individuals. EVs were isolated by ultrafiltration and characterized using transmission electron microscopy, tandem mass spectrometry (LC/MS/MS, and nanoparticle tracking analysis (NTA. Western blots confirmed the presence of HIV proteins. Gene ontology (GO analysis was performed using FunRich and HIV Human Interaction database (HHID. Results. EVs from urine were 30–400 nm in size. More EVs were in HIV+ patients, P<0.05, by NTA. HIV+ samples had 14,475 HPs using LC/MS/MS, while only 111 were in HIV−. HPs in the EVs were of exosomal origin. LC/MS/MS showed all HIV+ samples contained at least one HIV protein. GO analysis showed differences in proteins between HIV+ and HIV− samples and more than 50% of the published HPs in the HHID interacted with EV HIV proteins. Conclusion. Differences in the proteomic profile of EVs from HIV+ versus HIV− samples were found. HIV and HPs in EVs could be used to detect infection and/or diagnose HIV disease syndromes.

  14. Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles.

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    Sanada, Takahiro; Hirata, Yuichi; Naito, Yutaka; Yamamoto, Naoki; Kikkawa, Yoshiaki; Ishida, Yuji; Yamasaki, Chihiro; Tateno, Chise; Ochiya, Takahiro; Kohara, Michinori

    2017-03-01

    An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection. We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells). Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy. Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA-transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA-transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies. These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization-resistant route of HBV infection.

  15. Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

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    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  16. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

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    Luca Musante

    2015-01-01

    Full Text Available Diabetic nephropathy (DN is one of the major complications of diabetes mellitus (DM, leads to chronic kidney disease (CKD, and, ultimately, is the main cause for end-stage kidney disease (ESKD. Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  17. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

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    Jinghuan Li

    2015-05-01

    Full Text Available Extracellular vesicles (EVs play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors, some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

  18. Development and characterization of nanopore system for nano-vesicle analysis

    Science.gov (United States)

    Goyal, Gaurav

    Nano-vesicles have recently attracted a lot of attention in research and medical communities and are very promising next-generation drug delivery vehicles. This is due to their biocompatibility, biodegradability and their ability to protect drug cargo and deliver it to site-specific locations, while maintaining the desired pharmacokinetic profile. The interaction of these drug loaded vesicles with the recipient cells via adsorption, endocytosis or receptor mediated internalization involve significant bending and deformation and is governed by mechanical properties of the nano-vesicles. Currently, the mechanical characteristics of nano-vesicles are left unexplored because of the difficulties associated with vesicle analysis at sub-100 nm length scale. The need for a complete understanding of nano-vesicle interaction with each other and the recipient cells warrants development of an analytical tool capable of mechanical investigation of individual vesicles at sub-100 nm scale. This dissertation presents investigation of nano-vesicle deformability using resistive pulse sensing and solid-state nanopore devices. The dissertation is divided into four chapters. Chapter 1 discusses the motivation, specific aims and presents an overview of nanoparticle characterization techniques, resistive pulse sensing background and principles, techniques for fabricating solid-state nanopores, as well the deformation behavior of giant vesicles when placed in electric field. Chapter 2 is dedicated to understanding of the scientific principles governing transport of sub-100 nm particles in dilute solutions. We investigated the translocation of rigid nanoparticles through nanopores at salt concentrations exosomes derived from human breast cancer cell line. Exosomes also exhibit co-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

  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 <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the 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 <70 nm in suspension. Method: Urinary 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 (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section. Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.

  20. Maturation arrest of human oocytes at germinal vesicle stage

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    Zhi Qin Chen

    2010-01-01

    Full Text Available Maturation arrest of human oocytes may occur at various stages of the cell cycle. A total failure of human oocytes to complete meiosis is rarely observed during assisted conception cycles. We describe here a case of infertile couples for whom all oocytes repeatedly failed to mature at germinal vesicle (GV stage during in vitro fertilization/Intra cytoplasmic sperm injection (IVF/ICSI. The patient underwent controlled ovarian stimulation followed by oocyte retrieval and IVF/ICSI. The oocytes were stripped off cumulus cells prior to the ICSI procedure and their maturity status was defined. The oocyte maturation was repeatedly arrested at the GV. Oocyte maturation arrest may be the cause of infertility in this couple. The recognition of oocyte maturation arrest as a specific medical condition may contribute to the characterization of the currently known as "oocyte factor." The cellular and genetic mechanisms causing oocyte maturation arrest should be the subject for further investigation.

  1. Stem Cell Extracellular Vesicles: Extended Messages of Regeneration

    Science.gov (United States)

    Riazifar, Milad; Pone, Egest J.; Lötvall, Jan; Zhao, Weian

    2017-01-01

    Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications. PMID:27814025

  2. Giant plasma membrane vesicles: models for understanding membrane organization.

    Science.gov (United States)

    Levental, Kandice R; Levental, Ilya

    2015-01-01

    The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Spontaneous vesicle phase formation by pseudogemini surfactants in aqueous solutions.

    Science.gov (United States)

    Sun, Nan; Shi, Lijuan; Lu, Fei; Xie, Shuting; Zheng, Liqiang

    2014-08-14

    The phase behavior of a kind of pseudogemini surfactant in aqueous solutions, formed by the mixture of sodium dodecyl benzene sulfonate (SDBS) and butane-1,4-bis (methylimidazolium bromide) ([mim-C4-mim]Br2) or butane-1,4-bis(methylpyrrolidinium bromide) ([mpy-C4-mpy]Br2) in a molar ratio of 2 : 1, is reported in the present work. When [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 is mixed with SDBS in aqueous solutions, one cationic [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 molecule "bridges" two SDBS molecules by noncovalent interactions (e.g. electrostatic, π-π stacking, and σ-π interactions), behaving like a pseudogemini surfactant. Vesicles can be formed by this kind of pseudogemini surfactant, determined by freeze-fracture transmission electron microscopy (FF-TEM) or cryogenic-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). The mixed system of sodium dodecyl sulfate (SDS) with [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 was also constructed, and only micelles were observed. We infer that a pseudogemini surfactant is formed under the synergic effect of electrostatic, π-π stacking, and σ-π interactions in the SDBS/[mim-C4-mim]Br2/H2O system, while electrostatic attraction and hydrophobic interactions may provide the directional force for vesicle formation in the SDBS/[mpy-C4-mpy]Br2/H2O system.

  4. Development and dosimetric evaluation of radiochromic PCDA vesicle gel dosimeters

    International Nuclear Information System (INIS)

    Sun, P.; Fu, Y.C.; Hu, J.; Hao, N.; Huang, W.; Jiang, B.

    2016-01-01

    The gel dosimeter has the unique capacity in recording radiation dose distribution in three dimensions (3D), which has the specific advantages in dosimetry measurements where steep dose gradients exist, such as in intensity-modulated radiation therapy (IMRT), brachytherapy and so on. Some 3D dosimeters, such as Fricke gel dosimeters, polymer gel dosimeters, the PRESAGE plastic dosimeters and micelle gel dosimeters have appeared recently. However, there are several disadvantages of these 3D dosimeters limit their application in radiotherapy dose verification. In this study, a novel radiochromic gel dosimeter for 3D dose verification of radiotherapy was developed by dispersing nanovesicles self-assembled by 10,12-pentacosadiynoic acid (PCDA) into the tissue equivalence gel matrix. The characteristics of radiochromic PCDA vesicle gel dosimeters were evaluated. The results indicate that these radiochromic gel dosimeters have good linear dose response to X-ray irradiation in the dose range of 2–100 Gy. In addition, the radiochromic gel dosimeters breakthrough the limitations of the existing gel dosimeters such as diffusion effect, post-radiation effect, and poor forming ability. The response of the gel dosimeter does not show any dose rate dependence, energy dependence and temperature effect, and there was no obvious difference in the gel response between single and cumulative dose of fractional irradiation. Hence, the radiochromic PCDA vesicle gel dosimeters developed in this study could be generally applied to 3D dose verification in radiotherapy. - Highlights: • A novel radiochromic gel dosimeter was developed by dispersing PCDA nanovesicles into the tissue equivalence gel matrix. • This nanovesicle overcomes the dose image blurring caused by the diffusion of monomer molecules. • This nanovesicle limits the polymer chain growth, so as to reduce the post-radiation effect. • The gel matrixes possess excellent tissue equivalence and elastic strength, which

  5. Isolated galaxies

    International Nuclear Information System (INIS)

    Einasto, Maret

    1990-01-01

    To test for the possible presence of really isolated galaxies, which form a randomly distributed population in voids, we compare the distribution of most isolated galaxies in an observed sample with distributions of the same number of random points using the nearest neighbour test. The results show that the random population of really isolated galaxies does not exist - even the most isolated galaxies are connected with systems of galaxies, forming their outlying parts. (author)

  6. Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens.

    Science.gov (United States)

    Pors, Susanne E; Pedersen, Ida J; Skjerning, Ragnhild Bager; Thøfner, Ida C N; Persson, Gry; Bojesen, Anders M

    2016-11-15

    Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

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    Inés Lozano-Ramos

    2015-05-01

    Full Text Available Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9. The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm–Horsfall protein were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches.

  8. Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles.

    Science.gov (United States)

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; López-Ruano, Guillermo; Muntión, Sandra; Preciado, Silvia; Hernández-Ruano, Montserrat; Rosado, Belén; de las Heras, Natalia; Chillón, M Carmen; Hernández-Hernández, Ángel; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, Consuelo

    2015-08-01

    The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

    Science.gov (United States)

    Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

    2018-01-05

    Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally

  10. Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles

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    Chris Gardiner

    2014-11-01

    Full Text Available Introduction: Optical techniques are routinely used to size and count extracellular vesicles (EV. For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA provides both size and relative light scattering intensity (rLSI values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs. Methods: NTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values. Results: The following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364–1.375 and 1.398–1.414 for large placental EV (>200 nm. Large placental EV had a significantly higher RI than small placental EV (p1.40 were observed for some large (>200 nm microvesicles. Conclusion: This method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.

  11. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

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    Dorota Tataruch-Weinert

    2016-06-01

    Full Text Available Background: Urinary extracellular vesicles (UEVs represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods: UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results: The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters and chemical pretreatment (water purification tablet. For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b UEVs were enriched with small RNA, (c Ribosomal RNA peaks were not observed for any RNA extraction method used and (d RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion: Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA

  12. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Directory of Open Access Journals (Sweden)

    Daungruthai Jarukanont

    Full Text Available Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We

  13. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Science.gov (United States)

    Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

    2015-01-01

    Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

  14. Relationship of membrane-bound sulfhydryl groups to vitamin D-stimulated uptake of [75Se]Selenite by the brush border membrane vesicles from chick duodenum

    International Nuclear Information System (INIS)

    Mykkanen, H.M.; Wasserman, R.H.

    1990-01-01

    The uptake of selenite by purified brush border membrane vesicles isolated from duodena of rachitic or vitamin D-treated chicks was studied by using radioactive selenite and a rapid filtration technique. Cholecalciferol treatment (500 IU at 72 h) significantly enhanced selenite uptake, a response that decreased when the vesicles were stored at room temperature for 2.5 h prior to the uptake measurement. Preincubation of the vesicles in 1.0 mmol/L H2O2 reduced [75Se]selenite uptake, indicating the involvement of oxidizable groups in the uptake reaction. Iodoacetic acid (IAA), a sulfhydryl-blocking reagent, at 1-2 mmol/L concentration eliminated the difference in selenite uptake due to cholecalciferol and had no effect on vesicles from rachitic animals. A higher concentration of IAA (10 mmol/L) enhanced selenite uptake manyfold and increased the absolute difference due to cholecalciferol treatment. Single intravenous doses of 100 IU cholecalciferol, 100 IU ergocalciferol, or 0.1 micrograms 1,25-dihydroxycholecalciferol also stimulated selenite uptake, suggesting a general response to vitamin D compounds. Normal animals given a single dose of 1,25-dihydroxycholecalciferol 12 h prior to killing also responded. Treatments that enhanced the uptake of [75Se]selenite also increased the amount of membrane-bound sulfhydryl groups, suggesting the involvement of membrane-bound sulfhydryl groups in the vitamin D response. A significant increase in selenite uptake by intravenous 1,25-dihydroxycholecalciferol occurred within 10 min. This rapid effect provides a new tool to probe early biochemical effects of vitamin D on intestinal epithelium

  15. First report of detection of the putative receptor of Bacillus thuringiensis toxin Vip3Aa from black cutworm (Agrotis ipsilon

    Directory of Open Access Journals (Sweden)

    Gamal H. Osman

    2018-03-01

    Full Text Available Black cutworm (BCW Agrotis ipsilon, an economically important lepidopteran insect, has attracted a great attention. Bacillus thuringiensis (Bt is spore forming soil bacteria and is an excellent environment-friendly approach for the control of phytophagous and disease-transmitting insects. In fact, bio-pesticide formulations and insect resistant transgenic plants based on the bacterium Bt delta-endotoxin have attracted worldwide attention as a safer alternative to harmful chemical pesticides. The major objective of the current study was to understand the mechanism of interaction of Bt toxin with its receptor molecule(s. The investigation involved the isolation, identification, and characterization of a putative receptor – vip3Aa. In addition, the kinetics of vip toxin binding to its receptor molecule was also studied. The present data suggest that Vip3Aa toxin bound specifically with high affinity to a 48-kDa protein present at the brush border membrane vesicles (BBMV prepared from the midgut epithelial cells of BCW larvae. Keywords: Receptor, vip3Aa, Bacillus thuringiensis, BBMV

  16. EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Dae-Kyum Kim

    2013-03-01

    Full Text Available Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20–1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia (http://evpedia.info might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.

  17. Effect of Gamma Radiation on Amino Acid Based Vesicle Carrying Radiosensitizer

    International Nuclear Information System (INIS)

    Nur Ratasha Alia Mohd Rosli; Faizal Mohamed; Muhammad Amir Syafiq Mohd Sah; Irman Abdul Rahman

    2014-01-01

    Vesicles has been developed and studied to be used as a medium to transport radiosensitizer in treating cancer cells by increasing its sensitivity effectively towards the radiation given during radiotherapy. This study was conducted to investigate the effect of gamma radiation on amino acid-based vesicle carrying radiosensitizer. Amino acid based vesicles carrying radiosensitizer were synthesized using sonication method with sodium N-lauroylsarcosinate hydrate and decanol being the primary surfactant, while hydrogen peroxide and sodium hyaluronate as the encapsulated radiosensitizer. The synthesized vesicle was then irradiated at radiation doses equivalent to those given during radiotherapy. Irradiated vesicle carrying radiosensitizer were then characterized using Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR) and Polarized Light Microscope. Results obtained shows that there were no significant changes in morphology and molecular conformation of the synthesized vesicle after irradiation. Even at higher radiation dose of 100 Gray and 200 Gray, the results remained unchanged. This indicates that the synthesized vesicle carrying radiosensitizer is morphologically and spectroscopically stable even at high radiation doses. (author)

  18. An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

    International Nuclear Information System (INIS)

    Zhang, Xiaojun; Chen, Yuan; Chen, Yong

    2014-01-01

    Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release

  19. Squamous cell carcinoma of the seminal vesicle. Review of the related literature and case report

    Directory of Open Access Journals (Sweden)

    V. B. Matveev

    2015-01-01

    Full Text Available Seminal vesicle tumors are very rare malignancies which are not diagnosed in daily clinical oncology practice. Primary malignant tumors in seminal vesicle are difficult to define due to the lack of specific symptoms in the early stages of the disease. Another obstacle of proper diagnosis is the frequent invasion of tumors of the surrounding organs, especially the prostate, rectum and bladder which is difficult to differentiate. Very often seminal vesicle tumors are difficult to detect. Digital rectal examination as well as transrectal ultrasound scan (US could reveal a bulky mass in the retrovesical space. Computed tomography and magnetic resonance imaging (MRI are the main diagnostic methods which could help to reveal pathologic masses in the region of seminal vesicles. Levels of prostate-specific antigen, carcinoembryonic antigen and tumor markers specific for colorectal cancer are negative in seminal vesicle tumors.The world experience of treating seminal vesicle tumors is very limited. There is paucity of data regarding appropriate choice of surgical approach and further treatment strategy and most of the time the treatment is individualized and based on very scarce information. At the same time surgical approach may vary significantly from vesiculectomy to pelvic exenteration. Possibility of using any regimens of adjuvant radiation therapy, chemotherapy or hormone therapy is highly debatable. However, aggressive surgical approach with radical tumor removal followed by extended lymphodissection shows the most favorable results in survival of patients suffering from seminal vesicle cancer.Squamous cell carcinoma of the seminal vesicles is presumed to be an extremely rare disease as there are only 3 reports of it in the world literature. We report a case of patient B. suffering from squamous cell carcinoma of the right seminal vesicle whom we conducted an aggressive surgical approach – prostatovesiculectomy followed by resection of the

  20. A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations

    International Nuclear Information System (INIS)

    Simonsson, Carl; Madsen, Jakob Torp; Graneli, Annette; Andersen, Klaus E.; Karlberg, Ann-Therese; Jonsson, Charlotte A.; Ericson, Marica B.

    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 administered in micro- and nano-scale vesicle formulations. The sensitizing capacity of RBITC was studied using the murine local lymph node assay (LLNA) and the skin penetration properties were compared using diffusion cells in combination with two-photon microscopy (TPM). The lymph node cell proliferation, an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved penetration and increased formation of hapten-protein complexes in epidermis when RBITC is delivered in ethosomal formulations. - Graphical Abstract: Display Omitted

  1. Shear-induced formation of vesicles in membrane phases: Kinetics and size selection mechanisms, elasticity versus surface tension

    Science.gov (United States)

    Courbin, L.; Panizza, P.

    2004-02-01

    Multilamellar vesicles can be formed upon shearing lamellar phases (Lα) and phase-separated lamellar-sponge (Lα/L3) mixtures. In the first case, the vesicle volume fraction is always 100% and the vesicle size is monitored by elasticity (“onion textures”). In the second system the vesicle volume fraction can be tuned from 0 to 100% and the mean size results from a balance between capillary and viscous forces (“Taylor droplets”). However, despite these differences, in both systems we show that the formation of vesicles is a strain-controlled process monitored by a universal primary buckling instability of the lamellae.

  2. 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.

  3. Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE

    OpenAIRE

    Hu, Zonglin; Gogol, Edward P.; Lutkenhaus, Joe

    2002-01-01

    Selection of the division site in Escherichia coli is regulated by the min system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE. In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles. We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicle...

  4. Fusion of Sendai Virus with Vesicles of Oligomerizable Lipids: A Micro Calorimetric Analysis of Membrane Fusion

    OpenAIRE

    Ravoo, Bart Jan; Weringa, Wilke D.; Engberts, Jan B.F.N.

    2000-01-01

    Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4-(beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37°C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sen...

  5. Peripheral primitive neuroectodermal tumor of seminal vesicles: Is there a role for relatively aggressive treatment modalities?

    Directory of Open Access Journals (Sweden)

    Alessandro Crestani

    2014-12-01

    Full Text Available A 50 year old white man received an incidental ultrasound diagnosis of hypoechoic mass interesting the right seminal vesicle. A CT scan showed the presence of a 7.8 cm roundish cyst, originating from the right seminal vesicle. He had been followed by the removal of the right seminal vesicle and both the cystic lesion. The histological findings of the specimen documented the presence of small round cells compatible with Ewing’s sarcoma/PPNET. The patient received also adjuvant chemotherapy and radiation treatment. After 10 years, the follow-up is still negative.

  6. Unconditionally energy stable numerical schemes for phase-field vesicle membrane model

    Science.gov (United States)

    Guillén-González, F.; Tierra, G.

    2018-02-01

    Numerical schemes to simulate the deformation of vesicles membranes via minimizing the bending energy have been widely studied in recent times due to its connection with many biological motivated problems. In this work we propose a new unconditionally energy stable numerical scheme for a vesicle membrane model that satisfies exactly the conservation of volume constraint and penalizes the surface area constraint. Moreover, we extend these ideas to present an unconditionally energy stable splitting scheme decoupling the interaction of the vesicle with a surrounding fluid. Finally, the well behavior of the proposed schemes are illustrated through several computational experiments.

  7. 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

  8. Photoinduced bimolecular electron transfer kinetics in small unilamellar vesicles

    International Nuclear Information System (INIS)

    Choudhury, Sharmistha Dutta; Kumbhakar, Manoj; Nath, Sukhendu; Pal, Haridas

    2007-01-01

    Photoinduced electron transfer (ET) from N,N-dimethylaniline to some coumarin derivatives has been studied in small unilamellar vesicles (SUVs) of the phospholipid, DL-α-dimyristoyl-phosphatidylcholine, using steady-state and time-resolved fluorescence quenching, both below and above the phase transition temperature of the vesicles. The primary interest was to examine whether Marcus inversion [H. Sumi and R. A. Marcus, J. Chem. Phys. 84, 4894 (1986)] could be observed for the present ET systems in these organized assemblies. The influence of the topology of SUVs on the photophysical properties of the reactants and consequently on their ET kinetics has also been investigated. Absorption and fluorescence spectral data of the coumarins in SUVs and the variation of their fluorescence decays with temperature indicate that the dyes are localized in the bilayer of the SUVs. Time-resolved area normalized emission spectra analysis, however, reveals that the dyes are distributed in two different microenvironments in the SUVs, which we attribute to the two leaflets of the bilayer, one toward bulk water and the other toward the inner water pool. The microenvironments in the two leaflets are, however, not indicated to be that significantly different. Time-resolved anisotropy decays were biexponential for all the dyes in SUVs, and this has been interpreted in terms of the compound motion model according to which the dye molecules can experience a fast wobbling-in-cone type of motion as well as a slow overall rotating motion of the cone containing the molecule. The expected bimolecular diffusion-controlled rates in SUVs, as estimated by comparing the microviscosities in SUVs (determined from rotational correlation times) and that in acetonitrile solution, are much slower than the observed fluorescence quenching rates, suggesting that reactant diffusion (translational) does not play any role in the quenching kinetics in the present systems. Accordingly, clear inversions are

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

    International Nuclear Information System (INIS)

    Nieh, Mu-Ping; Dolinar, Paul; Kucerka, Norbert; Kline, Steven R.; Debeer-Schmitt, Lisa M.; Littrell, Ken; Katsaras, John

    2011-01-01

    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 applications that

  10. Morphological changes in vesicles and release of an encapsulated compound triggered by a photoresponsive Malachite Green leuconitrile derivative.

    Science.gov (United States)

    Uda, Ryoko M; Hiraishi, Eri; Ohnishi, Ryo; Nakahara, Yoshio; Kimura, Keiichi

    2010-04-20

    Photoinduced morphological changes in phosphatidylcholine vesicles are triggered by a Malachite Green leuconitrile derivative dissolved in the lipidic membrane, and are observed at Malachite Green derivative/lipid ratios Malachite Green derivative is a photoresponsive compound that undergoes ionization to afford a positive charge on the molecule by UV irradiation. The Malachite Green derivative exhibits amphiphilicity when ionized photochemically, whereas it behaves as a lipophilic compound under dark conditions. Cryo-transmission electron microscopy was used to determine vesicle morphology. The effects of the Malachite Green derivative on vesicles were studied by dynamic light scattering and fluorescence resonance energy transfer. Irradiation of vesicles containing the Malachite Green derivative induces nonspherical vesicle morphology, fusion of vesicles, and membrane solubilization, depending on conditions. Furthermore, irradiation of the Malachite Green derivative induces the release of a vesicle-encapsulated compound.

  11. Self-assembly of star micelle into vesicle in solvents of variable quality: the star micelle retains its core-shell nanostructure in the vesicle.

    Science.gov (United States)

    Liu, Nijuan; He, Qun; Bu, Weifeng

    2015-03-03

    Intra- and intermolecular interactions of star polymers in dilute solutions are of fundamental importance for both theoretical interest and hierarchical self-assembly into functional nanostructures. Here, star micelles with a polystyrene corona and a small ionic core bearing platinum(II) complexes have been regarded as a model of star polymers to mimic their intra- and interstar interactions and self-assembled behaviors in solvents of weakening quality. In the chloroform/methanol mixture solvents, the star micelles can self-assemble to form vesicles, in which the star micelles shrink significantly and are homogeneously distributed on the vesicle surface. Unlike the morphological evolution of conventional amphiphiles from micellar to vesicular, during which the amphiphilic molecules are commonly reorganized, the star micelles still retain their core-shell nanostructures in the vesicles and the coronal chains of the star micelle between the ionic cores are fully interpenetrated.

  12. Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

    Science.gov (United States)

    Herrera Sanchez, Maria Beatriz; Previdi, Sara; Bruno, Stefania; Fonsato, Valentina; Deregibus, Maria Chiara; Kholia, Sharad; Petrillo, Sara; Tolosano, Emanuela; Critelli, Rossana; Spada, Marco; Romagnoli, Renato; Salizzoni, Mauro; Tetta, Ciro; Camussi, Giovanni

    2017-07-27

    Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme

  13. Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease.

    Directory of Open Access Journals (Sweden)

    Davide Povero

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.Choline Deficient L-Amino Acid (CDAA and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.We observed statistically significant differences in the level of extracellular vesicles (EVs in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05, fibrosis (r2 = 0.66, p<0.05 and pathological angiogenesis (r2 = 0.71, p<0.05. Extensive characterization of blood EVs identified both microparticles (MPs and exosomes (EXO present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

  14. Structure of clathrin-coated vesicles from small-angle scattering experiments

    DEFF Research Database (Denmark)

    Pedersen, J.S.

    1993-01-01

    Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D2O solvent concentrations. The model...... used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. A constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein....... Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 x 10(6) Da. The coated vesicles consist, on average, of approximately 85% protein and 15% lipids. About 40% of the protein mass...

  15. Primary leiomyosarcoma of the seminal vesicle: Case report and review of the literature

    International Nuclear Information System (INIS)

    Cauvin, Cécile; Moureau-Zabotto, Laurence; Chetaille, Bruno; Hilgers, Werner; Denoux, Yves; Jacquemier, Jocelyne; Guiramand, Jérôme; Sarran, Anthony; Bertucci, François

    2011-01-01

    Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up

  16. Primary leiomyosarcoma of the seminal vesicle: case report and review of the literature.

    Science.gov (United States)

    Cauvin, Cécile; Moureau-Zabotto, Laurence; Chetaille, Bruno; Hilgers, Werner; Denoux, Yves; Jacquemier, Jocelyne; Guiramand, Jérôme; Sarran, Anthony; Bertucci, François

    2011-07-29

    Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up.

  17. Primary leiomyosarcoma of the seminal vesicle: Case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Guiramand Jérôme

    2011-07-01

    Full Text Available Abstract Background Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. Case Presentation We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. Conclusions We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up.

  18. Reactions of the hydrated electron with pyrene in lipid bilayer vesicles

    International Nuclear Information System (INIS)

    Schnecke, W.; Graetzel, M.; Henglein, A.

    1977-01-01

    Pyrene and some pyrene derivatives were solubilized in bilayer vesicles of lecithin and the rates of lecithin and the rates of reaction with the hydrated electron investigated. The concentration of the vesicles was 1.3 x 10 -7 M, that of pyrene 10 -6 - 10 -4 M. The rate constant decreases with increasing pyrene concentration. The effect is explained by the highly inhomogeneous distribution of pyrene molecules in the solutions. Only those pyrene molicules are reactive that reside close to the outer surface of the vesicles. The anions of pyrene formed disappear in a second order process. It is concluded that the anions are rapidly detached from their vesicular carriers and react with each other in the aqueous phase. Fluorescence, light scattering and electron microscopic investigations were also carried out to obtain information about the properties of the vesicles used. (orig.) [de

  19. Bile salt-induced cholesterol crystal formation from model bile vesicles: a time course study

    NARCIS (Netherlands)

    van de Heijning, B. J.; Stolk, M. F.; van Erpecum, K. J.; Renooij, W.; Groen, A. K.; vanBerge-Henegouwen, G. P.

    1994-01-01

    Precipitation of cholesterol crystals from vesicles is an important step in the pathogenesis of cholesterol gallstones. Little is known, however, about the kinetics and the mechanisms involved in cholesterol crystallization. Therefore, the time course of cholesterol crystal precipitation and lipid

  20. 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.

  1. The parachute morphology as equilibrium morphology of vesicle-polymer hybrids?

    NARCIS (Netherlands)

    Jung, M.; Hubert, D.H.W.; Herk, van A.M.; German, A.L.

    2000-01-01

    Polymerisation in vesicles leads to novel polymer colloid morphologies. Two morphologies are currently reported: the triple-shell and the parachute morphology. The termodynamic analysis of these two morphologies, presented here, stresses the importance of considering interfacial energies between

  2. Heterogeneous vesicles in mucous epithelial cells of posterior esophagus of Chinese giant salamander (Andrias davidianus

    Directory of Open Access Journals (Sweden)

    H. Zhang

    2015-08-01

    Full Text Available The Chinese giant salamander belongs to an old lineage of salamanders and endangered species. Many studies of breeding and disease regarding this amphibian had been implemented. However, the studies on the ultrastructure of this amphibian are rare. In this work, we provide a histological and ultrastructural investigation on posterior esophagus of Chinese giant salamander. The sections of amphibian esophagus were stained by hematoxylin & eosin (H&E. Moreover, the esophageal epithelium was observed by transmission electron microscopy (TEM. The results showed that esophageal epithelium was a single layer epithelium, which consisted of mucous cells and columnar cells. The esophageal glands were present in submucosa. The columnar cells were ciliated. According to the diverging ultrastructure of mucous vesicles, three types of mucous cells could be identified in the esophageal mucosa: i electron-lucent vesicles mucous cell (ELV-MC; ii electron-dense vesicles mucous cell (EDV-MC; and iii mixed vesicles mucous cell (MV-MC.

  3. Vesicle fusion observed by content transfer across a tethered lipid bilayer.

    Science.gov (United States)

    Rawle, Robert J; van Lengerich, Bettina; Chung, Minsub; Bendix, Poul Martin; Boxer, Steven G

    2011-10-19

    Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

    NARCIS (Netherlands)

    Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A; Del Portillo, Hernando A; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Görgens, André; Gupta, Ramesh C; Hendrix, An; Hermann, Dirk M; Hill, Andrew F; Hochberg, Fred; Horn, Peter A; de Kleijn, Dominique; Kordelas, Lambros; Kramer, Boris W; Krämer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J; Lim, Sai Kiang; Lötvall, Jan; Maguire, Casey A; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G; Rivera, Francisco J; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W M; Wauben, Marca|info:eu-repo/dai/nl/112675735; Andaloussi, Samir El; Théry, Clotilde; Rohde, Eva; Giebel, Bernd

    2015-01-01

    Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information

  5. 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.

  6. Heat Profiling of Three-Dimensionally Optically Trapped Gold Nanoparticles using Vesicle Cargo Release

    DEFF Research Database (Denmark)

    Kyrsting, Anders; Bendix, Pól Martin; Stamou, Dimitrios

    2011-01-01

    Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature...

  7. A practical guide to giant vesicles. Probing the membrane nanoregime via optical microscopy

    International Nuclear Information System (INIS)

    Dimova, Rumiana; Aranda, Said; Bezlyepkina, Natalya; Nikolov, Vesselin; Riske, Karin A; Lipowsky, Reinhard

    2006-01-01

    Research on giant vesicles is becoming increasingly popular. Giant vesicles provide model biomembrane systems for systematic measurements of mechanical and rheological properties of bilayers as a function of membrane composition and temperature, as well as hydrodynamic interactions. Membrane response to external factors (for example electric fields, ions and amphiphilic molecules) can be directly visualized under the microscope. In this paper we review our current understanding of lipid bilayers as obtained from studies on giant unilamellar vesicles. Because research on giant vesicles increasingly attracts the interest of scientists from various backgrounds, we also try to provide a concise introduction for newcomers in the field. Finally, we summarize some recent developments on curvature effects induced by polymers, domain formation in membranes and shape transitions induced by electric fields

  8. Small unilamellar vesicles as reagents: a chemically defined, quantitative assay for lectins

    Energy Technology Data Exchange (ETDEWEB)

    Rando, R.R.

    1981-01-01

    Samll unilamellar vesicles containing synthetic glycolipids can be prepared. These vesicles are aggregated by the appropriate lectin (Orr et al., 1979; Rando and Bangerter, 1979; Slama and Rando, 1980). It is shown here that extent of aggregation of these vesicles as measured by light scattering at 360 nm, is, under certain conditions, linear with amount of lectin added. This forms the basis of a rapid and simple quantitative assay for lectins using the modified vesicles as a defined chemical substrate. The assay is sensitive to lectin concentrations in the low ..mu..g range. The assay is applied here to studies on concanavalin A, Ricinus communis agglutinin and the ..cap alpha..-fucosyl binding lectin from Ulex europaeus (Type I).

  9. Statistical Modelling of Synaptic Vesicles Distribution and Analysing their Physical Characteristics

    DEFF Research Database (Denmark)

    Khanmohammadi, Mahdieh

    transmission electron microscopy is used to acquire images from two experimental groups of rats: 1) rats subjected to a behavioral model of stress and 2) rats subjected to sham stress as the control group. The synaptic vesicle distribution and interactions are modeled by employing a point process approach......This Ph.D. thesis deals with mathematical and statistical modeling of synaptic vesicle distribution, shape, orientation and interactions. The first major part of this thesis treats the problem of determining the effect of stress on synaptic vesicle distribution and interactions. Serial section...... on differences of statistical measures in section and the same measures in between sections. Three-dimensional (3D) datasets are reconstructed by using image registration techniques and estimated thicknesses. We distinguish the effect of stress by estimating the synaptic vesicle densities and modeling...

  10. Meningococcal outer membrane vesicle composition-dependent activation of the innate immune response

    NARCIS (Netherlands)

    Zariri, Afshin; Beskers, Joep; van de Waterbeemd, Bas; Hamstra, Hendrik Jan; Bindels, Tim H E; van Riet, Elly; van Putten, Jos P M; van der Ley, Peter

    2016-01-01

    Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen associated molecular patterns including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an

  11. A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing

    NARCIS (Netherlands)

    Vogel, Robert; Coumans, Frank A. W.; Maltesen, Raluca G.; Böing, Anita N.; Bonnington, Katherine E.; Broekman, Marike L.; Broom, Murray F.; Buzás, Edit I.; Christiansen, Gunna; Hajji, Najat; Kristensen, Søren R.; Kuehn, Meta J.; Lund, Sigrid M.; Maas, Sybren L. N.; Nieuwland, Rienk; Osteikoetxea, Xabier; Schnoor, Rosalie; Scicluna, Benjamin J.; Shambrook, Mitch; de Vrij, Jeroen; Mann, Stephen I.; Hill, Andrew F.; Pedersen, Shona

    2016-01-01

    Background: Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several

  12. Next-generation outer membrane vesicle vaccines from concept to clinical trials

    NARCIS (Netherlands)

    Waterbeemd, van de B.

    2013-01-01

    Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide

  13. Quantitative Proteomics Reveals Distinct Differences in the Protein Content of Outer Membrane Vesicle Vaccines

    NARCIS (Netherlands)

    Waterbeemd, van de B.; Mommen, G.P.M.; Pennings, J.L.A.; Eppink, M.H.M.; Wijffels, R.H.; Pol, van der L.A.; Jong, de A.P.J.M.

    2013-01-01

    At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in

  14. Concise review : Developing best-practice models for the therapeutic use of extracellular vesicles

    NARCIS (Netherlands)

    Reiner, Agnes T.; Witwer, Kenneth W.; Van Balkom, Bas W.M.; De Beer, Joel; Brodie, Chaya; Corteling, Randolph L.; Gabrielsson, Susanne; Gimona, Mario; Ibrahim, Ahmed G.; De Kleijn, Dominique; Lai, Charles P.; Tvall, Jan Lo; Del Portillo, Hernando A; Reischl, Ilona G; Riazifar, Milad; Salomon, Carlos; Tahara, Hidetoshi; Toh, Wei Seong; Wauben, Marca H M; Yang, Vicky K.; Yang, Yijun; Yeo, Ronne Wee Yeh; Yin, Hang; Giebel, Bernd; Rohde, Eva; Lim, Sai Kiang

    2017-01-01

    Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular

  15. The role of extracellular vesicles when innate meets adaptive.

    Science.gov (United States)

    Groot Kormelink, Tom; Mol, Sanne; de Jong, Esther C; Wauben, Marca H M

    2018-04-03

    Innate immune cells are recognized for their rapid and critical contribution to the body's first line of defense against invading pathogens and harmful agents. These actions can be further amplified by specific adaptive immune responses adapted to the activating stimulus. Recently, the awareness has grown that virtually all innate immune cells, i.e., mast cells, neutrophils, macrophages, eosinophils, basophils, and NK cells, are able to communicate with dendritic cells (DCs) and/or T and B cells, and thereby significantly contribute to the orchestration of adaptive immune responses. The means of communication that are thus far primarily associated with this function are cell-cell contacts and the release of a broad range of soluble mediators. Moreover, the possible contribution of innate immune cell-derived extracellular vesicles (EVs) to the modulation of adaptive immunity will be outlined in this review. EVs are submicron particles composed of a lipid bilayer, proteins, and nucleic acids released by cells in a regulated fashion. EVs are involved in intercellular communication between multiple cell types, including those of the immune system. A good understanding of the mechanisms by which innate immune cell-derived EVs influence adaptive immune responses, or vice versa, may reveal novel insights in the regulation of the immune system and can open up new possibilities for EVs (or their components) in controlling immune responses, either as a therapy, target, or as an adjuvant in future immune modulating treatments.

  16. EVpedia: a community web portal for extracellular vesicles research.

    Science.gov (United States)

    Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W M; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E; Buée, Luc; Buzás, Edit I; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S; Desiderio, Dominic M; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M; Gardiner, Chris; Giebel, Bernd; Greening, David W; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F; Hill, Michelle M; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; Del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I; Rodrigues, Marcio L; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

    2015-03-15

    Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

    Directory of Open Access Journals (Sweden)

    Stefanie Fischer

    Full Text Available The biological relevance of extracellular vesicles (EV in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

  18. Vesicle Origami and the Influence of Cholesterol on Lipid Packing.

    Science.gov (United States)

    Tanasescu, Radu; Lanz, Martin A; Mueller, Dennis; Tassler, Stephanie; Ishikawa, Takashi; Reiter, Renate; Brezesinski, Gerald; Zumbuehl, Andreas

    2016-05-17

    The artificial phospholipid Pad-PC-Pad was analyzed in 2D (monolayers at the air/water interface) and 3D (aqueous lipid dispersions) systems. In the gel phase, the two leaflets of a Pad-PC-Pad bilayer interdigitate completely, and the hydrophobic bilayer region has a thickness comparable to the length of a single phospholipid acyl chain. This leads to a stiff membrane with no spontaneous curvature. Forced into a vesicular structure, Pad-PC-Pad has faceted geometry, and in its extreme form, tetrahedral vesicles were found as predicted a decade ago. Above the main transition temperature, a noninterdigitated Lα phase with fluid chains has been observed. The addition of cholesterol leads to a slight decrease of the main transition temperature and a gradual decrease in the transition enthalpy until the transition vanishes at 40 mol % cholesterol in the mixture. Additionally, cholesterol pulls the chains apart, and a noninterdigitated gel phase is observed. In monolayers, cholesterol has an ordering effect on liquid-expanded phases and disorders condensed phases. The wavenumbers of the methylene stretching vibration indicate the formation of a liquid-ordered phase in mixtures with 40 mol % cholesterol.

  19. Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Yuana Yuana

    2017-07-01

    Full Text Available Microbubbles-assisted ultrasound (USMB has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs. USMB was performed on a monolayer of head-and-neck cancer cells (FaDu with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30–1200 nm. Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.

  20. Biological properties of extracellular vesicles and their physiological functions

    Science.gov (United States)

    Yáñez-Mó, María; Siljander, Pia R.-M.; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E.; Buzas, Edit I.; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Silva, Anabela Cordeiro-da; Fais, Stefano; Falcon-Perez, Juan M.; Ghobrial, Irene M.; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H. H.; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Hoen, Esther N.M. Nolte-‘t; Nyman, Tuula A.; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; del Portillo, Hernando A.; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N.; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem; Stukelj, Roman; Van der Grein, Susanne G.; Vasconcelos, M. Helena; Wauben, Marca H. M.; De Wever, Olivier

    2015-01-01

    In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system. PMID:25979354

  1. The key role of extracellular vesicles in the metastatic process.

    Science.gov (United States)

    Zhao, Hongyun; Achreja, Abhinav; Iessi, Elisabetta; Logozzi, Mariantonia; Mizzoni, Davide; Di Raimo, Rossella; Nagrath, Deepak; Fais, Stefano

    2018-01-01

    Extracellular vesicles (EVs), including exosomes, have a key role in the paracrine communication between organs and compartments. EVs shuttle virtually all types of biomolecules such as proteins, lipids, nucleic acids, metabolites and even pharmacological compounds. Their ability to transfer their biomolecular cargo into target cells enables EVs to play a key role in intercellular communication that can regulate cellular functions such as proliferation, apoptosis and migration. This has led to the emergence of EVs as a key player in tumor growth and metastasis through the formation of "tumor niches" in target organs. Recent data have also been shown that EVs may transform the microenvironment of primary tumors thus favoring the selection of cancer cells with a metastatic behavior. The release of EVs from resident non-malignant cells may contribute to the metastatic processes as well. However, cancer EVs may induce malignant transformation in resident mesenchymal stem cells, suggesting that the metastatic process is not exclusively due to circulating tumor cells. In this review, we outline and discuss evidence-based roles of EVs in actively regulating multiple steps of the metastatic process and how we can leverage EVs to impair metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Biological properties of extracellular vesicles and their physiological functions

    Directory of Open Access Journals (Sweden)

    María Yáñez-Mó

    2015-05-01

    Full Text Available In the past decade, extracellular vesicles (EVs have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

  3. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  4. Impact of lysosome status on extracellular vesicle content and release.

    Science.gov (United States)

    Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P

    2016-12-01

    Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. Copyright © 2016. Published by Elsevier B.V.

  5. Extracellular membrane vesicles and immune regulation in the brain

    Directory of Open Access Journals (Sweden)

    Stefano ePluchino

    2012-05-01

    Full Text Available The brain is characterized by a complex and integrated network of interacting cells in which cell-to-cell communication is critical for proper development and function. Initially considered as an immune privileged site, the brain is now regarded as an immune specialized system. Accumulating evidence reveals the presence of immune components in the brain, as well as extensive bidirectional communication that takes place between the nervous and the immune system both under homeostatic and pathological conditions. In recent years the secretion of extracellular membrane vesicles (EMVs has been described as a new and evolutionary well-conserved mechanism of cell-to-cell communication, with EMVs influencing the microenvironment through the traffic of bioactive molecules that include proteins and nucleic acids, such as DNA, protein coding and non coding RNAs. Increasing evidence suggests that EMVs are a promising candidate to study cross-boundary cell-to-cell communication pathways. Herein we review the role of EMVs secreted by neural cells in modulating the immune response(s within the brain under physiological and pathological circumstances.

  6. Staphylococcus aureus ?-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

    OpenAIRE

    Thay, Bernard; Wai, Sun Nyunt; Oscarsson, Jan

    2013-01-01

    Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol...

  7. Vesicle fluctuation analysis of the effects of sterols on membrane bending rigidity

    DEFF Research Database (Denmark)

    Henriksen, Jonas Rosager; Rowat, Amy C.; Ipsen, John H.

    2004-01-01

    Sterols are regulators of both biological function and structure. The role of cholesterol in promoting the structural and mechanical stability of membranes is widely recognized. Knowledge of how the related sterols, lanosterol and ergosterol, affect membrane mechanical properties is sparse. This ...... on vesicle behaviour are also discussed. These recent modifications render vesicle fluctuation analysis an efficient and accurate method for determining how cholesterol, lanosterol, and ergosterol increase membrane bending rigidity....

  8. 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

    Quantitative characterization of the lateral structure of curved membranes based on fluorescence microscopy requires knowledge of the fluorophore distribution on the surface. We present an image analysis approach for extraction of the fluorophore distribution on a spherical lipid vesicle from...... 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...

  9. Penetration enhancer-containing vesicles (PEVs) as carriers for cutaneous delivery of minoxidil.

    Science.gov (United States)

    Mura, Simona; Manconi, Maria; Sinico, Chiara; Valenti, Donatella; Fadda, Anna Maria

    2009-10-01

    The aim of this work was to evaluate the ability of a few different penetration enhancers to produce elastic vesicles with soy lecithin and the influence of the obtained vesicles on in vitro (trans)dermal delivery of minoxidil. To this purpose, so-called Penetration Enhancer-containing Vesicles (PEVs) were prepared as dehydrated-rehydrated vesicles by using soy lecithin and different amounts of three penetration enhancers, 2-(2-ethoxyethoxy)ethanol (Transcutol), capryl-caproyl macrogol 8-glyceride (Labrasol), and cineole. Soy lecithin liposomes, without penetration enhancers, were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, and vesicle deformability. The influence of PEVs on (trans)dermal delivery of minoxidil was studied by in vitro diffusion experiments through newborn pig skin in comparison with traditional liposomes and ethanolic solutions of the drug also containing each penetration enhancer. A skin pre-treatment study using empty PEVs and conventional liposomes was also carried out. Results showed that all the used penetration enhancers were able to give more deformable vesicles than conventional liposomes with a good drug entrapment efficiency and stability. In vitro skin penetration data showed that PEVs were able to give a statistically significant improvement of minoxidil deposition in the skin in comparison with classic liposomes and penetration enhancer-containing drug ethanolic solutions without any transdermal delivery. Moreover, the most deformable PEVs, prepared with Labrasol and cineole, were also able to deliver to the skin a higher total amount of minoxidil than the PE alcoholic solutions thus suggesting that minoxidil delivery to the skin was strictly correlated to vesicle deformability, and therefore to vesicle composition.

  10. Kadar Prostaglandin F2? pada Cairan Vesikula Seminalis dan Produk Sel Monolayer Vesikula Seminalis Sapi Bali (CONCENTRATIONS OF PROSTAGLANDIN F2? IN SEMINAL VESICLE FLUID AND PRODUCT OF SEMINAL VESICLE MONOLAYER CELLS OF BALI CATTLE

    Directory of Open Access Journals (Sweden)

    Tjok Gde Oka Pemayun

    2007-12-01

    Full Text Available In this study, the concentration of prostaglandin F2 ? (PGF2? in seminal vesicle fluid and seminal vesicle monolayer cell cultures of Bali cattle was determined. The seminal vesicle fluid was aspirated and the epithelial cells of the seminal vesicles were cultured in tissue culture medium (TCM 199 growth medium containing 10% fetal calf serum (FCS and 10% oestrus mares serum (EMS with a density of 1.9 x 106 cells / ml medium. Following an incubation at 38.50 C in 5% CO2 atmosphere for 6 days and the level of PGF2 ? in the original seminal vesicle fluid and in the cell culture medium were determined by radioimmunoassay techniques (RIA. The results showed that the level of PGF2 ? in the non-extracted monolayer culture of seminal vesicle (1287,50 ± 3,39 pg/ml was significantly higher than that of detected in non-extracted seminal vesicle fluid (1,23 ± 0,79 pg/ml. In contrast, after extraction the level of PGF2 ? in seminal vesicle monolayer cell cultures (218,33 ± 2,87 pg/ml significantly decreased as compared to seminal vesicle fluid (1750,83 ± 2,71 pg/ml. In conclusion the highest level of PGF2 ? was found in the extract of seminal vesicle fluid.

  11. Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

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    Cuc Quynh Nguyen Truong

    2014-11-01

    Full Text Available Priming of secretory vesicles is a prerequisite for their Ca2+-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca2+-dependent activator protein for secretion (CAPS also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

  12. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Science.gov (United States)

    Briers, Yves; Staubli, Titu; Schmid, Markus C; Wagner, Michael; Schuppler, Markus; Loessner, Martin J

    2012-01-01

    Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  13. The Function of Gas Vesicles in Halophilic Archaeaand Bacteria: Theories and Experimental Evidence

    Science.gov (United States)

    Oren, Aharon

    2012-01-01

    A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms. PMID:25371329

  14. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Directory of Open Access Journals (Sweden)

    Yves Briers

    Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  15. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

    Science.gov (United States)

    Moon, James J.; Suh, Heikyung; Bershteyn, Anna; Stephan, Matthias T.; Liu, Haipeng; Huang, Bonnie; Sohail, Mashaal; Luo, Samantha; Ho Um, Soong; Khant, Htet; Goodwin, Jessica T.; Ramos, Jenelyn; Chiu, Wah; Irvine, Darrell J.

    2011-03-01

    Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

  16. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

    Science.gov (United States)

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2015-10-08

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

  17. Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

    Science.gov (United States)

    El Harane, Nadia; Kervadec, Anaïs; Bellamy, Valérie; Pidial, Laetitia; Neametalla, Hany J; Perier, Marie-Cécile; Lima Correa, Bruna; Thiébault, Léa; Cagnard, Nicolas; Duché, Angéline; Brunaud, Camille; Lemitre, Mathilde; Gauthier, Jeanne; Bourdillon, Alexandra T; Renault, Marc P; Hovhannisyan, Yeranuhi; Paiva, Solenne; Colas, Alexandre R; Agbulut, Onnik; Hagège, Albert; Silvestre, Jean-Sébastien; Menasché, Philippe; Renault, Nisa K E

    2018-05-21

    We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left

  18. Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis

    International Nuclear Information System (INIS)

    Nakadera, Eisuke; Yamashina, Shunhei; Izumi, Kousuke; Inami, Yoshihiro; Sato, Toshifumi; Fukushima, Hirofumi; Kon, Kazuyoshi; Ikejima, Kenichi; Ueno, Takashi; Watanabe, Sumio

    2016-01-01

    Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD. - Highlights: • Hepatic steatosis causes accumulation of autophagic vesicles in hepatocytes. • Hepatic steatosis disturbs

  19. Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Nakadera, Eisuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke; Inami, Yoshihiro; Sato, Toshifumi; Fukushima, Hirofumi; Kon, Kazuyoshi; Ikejima, Kenichi [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Division of Proteomics and Biomolecular Science, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2016-01-22

    Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD. - Highlights: • Hepatic steatosis causes accumulation of autophagic vesicles in hepatocytes. • Hepatic steatosis disturbs

  20. Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

    Directory of Open Access Journals (Sweden)

    Sander A. A. Kooijmans

    2016-03-01

    Full Text Available Background: Extracellular vesicles (EVs are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI anchor signal peptides derived from decay-accelerating factor (DAF. EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results: V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression, but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

  1. Extracellular vesicles have variable dose-dependent effects on cultured draining cells in the eye.

    Science.gov (United States)

    Tabak, Saray; Schreiber-Avissar, Sofia; Beit-Yannai, Elie

    2018-03-01

    The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non-pigmented ciliary epithelium (NPCE)-derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose-response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE-derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β-Catenin, Axin2 and LEF1) and protein (β-Catenin, GSK-3β) expression using real-time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β-Catenin, GSK-3β, as opposed to exposure to high exosomal concentrations. Pro-MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE- or RPE-derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration-dependent at their target site. Specifically in the present study, we described a general dose-response at the gene and MMPs activity and a different dose-response regarding key canonical Wnt proteins expression. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. Increased Urinary Extracellular Vesicle Sodium Transporters in Cushing's Syndrome with Hypertension.

    Science.gov (United States)

    Salih, Mahdi; Bovée, Dominique M; van der Lubbe, Nils; Danser, Alexander H J; Zietse, Robert; Feelders, Richard A; Hoorn, Ewout J

    2018-05-02

    Increased renal sodium reabsorption contributes to hypertension in Cushing's syndrome (CS). Renal sodium transporters can be analyzed non-invasively in urinary extracellular vesicles (uEVs). To analyze renal sodium transporters in uEVs of patients with CS and hypertension. Observational study. University hospital. uEVs were isolated by ultracentrifugation and analyzed by immunoblotting in 10 CS patients and 7 age-matched healthy subjects. In 7 CS patients uEVs were analyzed before and after treatment. uEV protein abundance. The 10 CS patients were divided in those with suppressed and non-suppressed renin-angiotensin-aldosterone system (RAAS, n = 5/group). CS patients with suppressed RAAS had similar blood pressure but significantly lower serum potassium than CS patients with non-suppressed RAAS. Compared to healthy subjects, only those with suppressed RAAS had higher phosphorylated Na+-K+-Cl- cotransporter type 2 (pNKCC2) and higher total and phosphorylated Na+-Cl- cotransporter (NCC) in uEVs. Serum potassium but not urinary free cortisol correlated with pNKCC2, pNCC, and NCC in uEVs. Treatment of CS reversed the increases in pNKCC2, NCC, and pNCC. CS increases renal sodium transporter abundance in uEVs especially in patients with hypertension and suppressed RAAS. As potassium has recently been identified as an important driver of NCC activity, low serum potassium may also contribute to increased renal sodium reabsorption and hypertension in CS. These results may also be relevant for hypertension induced by exogenous glucocorticoids.

  3. Transdermal delivery of flurbiprofen from surfactant-based vesicles: particle characterization and the effect of water on in vitro transport.

    Science.gov (United States)

    Uchino, Tomonobu; Matsumoto, Yuiko; Murata, Akiko; Oka, Toshihiko; Miyazaki, Yasunori; Kagawa, Yoshiyuki

    2014-04-10

    Flurbiprofen loaded rigid and elastic vesicles comprising the bilayer-forming surfactant sucrose-ester laurate were prepared by the film rehydration and extrusion method. The charge-inducing agent sodium dodecyl sulfate, and the micelle-forming surfactants, sorbitan monolaurate, polyethylene glycol monolaurate, and polysorbate 20, were used to enhance elasticity. Vesicle formulations were evaluated for size, zeta potential, (1)H and (19)F nuclear magnetic resonance (NMR) spectra, and in vitro skin permeation across Yucatan micropig (YMP) skin. Vesicle formulations were stable for 2 weeks and their mean sizes were 95-135 nm. NMR spectroscopy showed that flurbiprofen molecular mobility was restricted by interaction with vesicle components because of entrapment in vesicle bilayers. Moreover, sorbitan monolaurate-containing vesicles strongly retained flurbiprofen molecules. After non-occlusive application to YMP skin, flurbiprofen transport from all vesicle formulations was superior to that of flurbiprofen alone and remarkably decreased after water vaporization. Polarization microscopy and small-angle X-ray diffraction analysis showed that the vesicle formulation was transferred to liquid crystalline state. Suppression of vesicle transition to the liquid crystalline state was observed with applications of both large quantities and diluted samples. The presence of water in the formulations was associated with maintenance of the vesicle structure and greater flurbiprofen transport across YMP skin. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006.

    Science.gov (United States)

    Monson, Rita E; Tashiro, Yosuke; Salmond, George P C

    2016-09-01

    Gas vesicles are intracellular proteinaceous organelles that facilitate bacterial colonization of static water columns. In the enterobacterium Serratia sp. ATCC 39006, gas vesicle formation requires the proteins GvpA1, GvpF1, GvpG, GvpA2, GvpK, GvpA3, GvpF2 and GvpF3 and the three gas vesicle regulatory proteins GvrA, GvrB and GvrC. Deletion of gvpC alters gas vesicle robustness and deletion of gvpN or gvpV results in small bicone vesicles. In this work, we assessed the impacts on gas vesicle formation when each of these 14 essential proteins was overexpressed. Overproduction of GvpF1, GvpF2, GvrA, GvrB or GvrC all resulted in significantly reduced gas vesicle synthesis. Perturbations in gas vesicle formation were also observed when GvpV and GvpA3 were in excess. In addition to impacts on gas vesicle formation, overproduction of GvrA or GvrB led to elevated biosynthesis of the tripyrrole pigment, prodigiosin, a secondary metabolite of increasing medical interest due to its antimalarial and anticancer properties. Finally, when GvpG was overexpressed, gas vesicles were still produced, but the cells exhibited a growth defect. Further analysis showed that induction of GvpG arrested cell growth and caused a drop in viable count, suggesting a possible physiological role for this protein linking gas vesicle biogenesis and binary fission. These combined results demonstrate that the stoichiometry of individual gas vesicle proteins is crucially important for controlled organelle morphogenesis and flotation and provides evidence for the first link between gas vesicle assembly and cell division, to our knowledge.

  5. Radical prostatectomy, sparing of the seminal vesicles, and painful orgasm.

    Science.gov (United States)

    Mogorovich, Andrea; Nilsson, Andreas E; Tyritzis, Stavros I; Carlsson, Stefan; Jonsson, Martin; Haendler, Leif; Nyberg, Tommy; Steineck, Gunnar; Wiklund, N Peter

    2013-05-01

    Erectile dysfunction has been widely investigated as the major factor responsible for sexual bother in patients after radical prostatectomy (RP); painful orgasm (PO) is one element of this bother, but little is known about its prevalence and its effects on sexual health. This study aims to investigate the prevalence of PO and to identify potential risk factors. A total of 1,411 consecutive patients underwent open (radical retropubic prostatectomy) or robot-assisted laparoscopic RP between 2002 and 2006. The patients were asked to complete a study-specific questionnaire. Of a total of 145 questions, 5 dealt with the orgasmic characteristics. The questionnaire was also administered to a comparison group of 442 persons, matched for age and area of residency. The response rate was 91% (1,288 patients). A total of 143 (11%) patients reported PO. Among the 834 men being able to have an orgasm, the prevalence was 18% vs. 6% in the comparison group (relative risk [RR] 2.8, 95% confidence interval [CI] 1.7-4.5). When analyzed as independent variables, bilateral seminal vesicle (SV)-sparing approach (RR 2.33, 95% CI 1.0-5.3, P = 0.045) and age <60 years were significantly related to the presence of PO (95% CI 0.5-0.9, P = 0.019). After adjustment for age, bilateral SV-sparing still remained a significant predictor for occurrence of PO. We found that PO occurs significantly more often in patients undergoing bilateral SV-sparing RP when compared with age-matched comparison population. © 2013 International Society for Sexual Medicine.

  6. Extracellular vesicle communication pathways as regulatory targets of oncogenic transformation.

    Science.gov (United States)

    Choi, Dongsic; Lee, Tae Hoon; Spinelli, Cristiana; Chennakrishnaiah, Shilpa; D'Asti, Esterina; Rak, Janusz

    2017-07-01

    Pathogenesis of human cancers bridges intracellular oncogenic driver events and their impact on intercellular communication. Among multiple mediators of this 'pathological connectivity' the role of extracellular vesicles (EVs) and their subsets (exosomes, ectosomes, oncosomes) is of particular interest for several reasons. The release of EVs from cancer cells represents a unique mechanism of regulated expulsion of bioactive molecules, a process that also mediates cell-to-cell transfer of lipids, proteins, and nucleic acids. Biological effects of these processes have been implicated in several aspects of cancer-related pathology, including tumour growth, invasion, angiogenesis, metastasis, immunity and thrombosis. Notably, the emerging evidence suggests that oncogenic mutations may impact several aspects of EV-mediated cell-cell communication including: (i) EV release rate and protein content; (ii) molecular composition of cancer EVs; (iii) the inclusion of oncogenic and mutant macromolecules in the EV cargo; (iv) EV-mediated release of genomic DNA; (v) deregulation of mechanisms responsible for EV biogenesis (vesiculome) and (vi) mechanisms of EV uptake by cancer cells. Intriguingly, EV-mediated intercellular transfer of mutant and oncogenic molecules between subpopulations of cancer cells, their indolent counterparts and stroma may exert profound biological effects that often resemble (but are not tantamount to) oncogenic transformation, including changes in cell growth, clonogenicity and angiogenic phenotype, or cause cell stress and death. However, several biological barriers likely curtail a permanent horizontal transformation of normal cells through EV-mediated mechanisms. The ongoing analysis and targeting of EV-mediated intercellular communication pathways can be viewed as a new therapeutic paradigm in cancer, while the analysis of oncogenic cargo contained in EVs released from cancer cells into biofluids is being developed for clinical use as a biomarker

  7. Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

    Science.gov (United States)

    Gui, M J; Dashper, S G; Slakeski, N; Chen, Y-Y; Reynolds, E C

    2016-10-01

    Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Quantification of Prostate and Seminal Vesicle Interfraction Variation During IMRT

    International Nuclear Information System (INIS)

    Frank, Steven J.; Dong Lei; Kudchadker, Rajat J.; De Crevoisier, Renaud; Lee, Andrew K.; Cheung, Rex; Choi, Seungtaek; O'Daniel, Jennifer; Tucker, Susan L.; Wang He; Kuban, Deborah A.

    2008-01-01

    Purpose: To quantify the interfraction variability in prostate and seminal vesicle (SV) positions during a course of intensity-modulated radiotherapy (IMRT) using an integrated computed tomography (CT)-linear accelerator system and to assess the impact of rectal and bladder volume changes. Methods and Materials: We studied 15 patients who had undergone IMRT for prostate carcinoma. Patients had one pretreatment planning CT scan followed by three in-room CT scans per week using a CT-on-rails system. The prostate, bladder, rectum, and pelvic bony anatomy were contoured in 369 CT scans. Using the planning CT scan as a reference, the volumetric and positional changes were analyzed in the subsequent CT scans. Results: For all 15 patients, the mean systematic internal prostate and SV variation was 0.1 ± 4.1 mm and 1.2 ± 7.3 mm in the anteroposterior axis, -0.5 ± 2.9 mm and -0.7 ± 4.5 mm in the superoinferior axis, and 0.2 ± 0.9 mm and -0.9 ± 1.9 mm in the lateral axis, respectively. The mean magnitude of the three-dimensional displacement vector was 4.6 ± 3.5 mm for the prostate and 7.6 ± 4.7 mm for the SVs. The rectal and bladder volume changes during treatment correlated with the anterior and superior displacement of the prostate and SVs. Conclusion: The dominant prostate and SV variations occurred in the anteroposterior and superoinferior directions. The systematic prostate and SV variation between the treatment planning CT and daily therapy as a result of the rectal and bladder volume changes emphasizes the need for daily directed target localization and/or immobilization techniques

  9. Astrocytic Pathological Calcium Homeostasis and Impaired Vesicle Trafficking in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Nina Vardjan

    2017-02-01

    Full Text Available Although the central nervous system (CNS consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer’s disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer’s disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration.

  10. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

    Science.gov (United States)

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

    2016-11-01

    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

  11. Placental Nano-vesicles Target to Specific Organs and Modulate Vascular Tone In Vivo.

    Science.gov (United States)

    Tong, Mancy; Stanley, Joanna L; Chen, Q; James, Joanna L; Stone, Peter R; Chamley, Larry W

    2017-11-01

    How do nano-vesicles extruded from normal first trimester human placentae affect maternal vascular function? Placental nano-vesicles affect the ability of systemic mesenteric arteries to undergo endothelium- and nitric oxide- (NO-) dependent vasodilation in vivo in pregnant mice. Dramatic cardiovascular adaptations occur during human pregnancy, including a substantial decrease in total peripheral resistance in the first trimester. The human placenta constantly extrudes extracellular vesicles that can enter the maternal circulation and these vesicles may play an important role in feto-maternal communication. Human placental nano-vesicles were administered into CD1 mice via a tail vein and their localization and vascular effects at 30 min and 24 h post-injection were investigated. Nano-vesicles from normal first trimester human placentae were collected and administered into pregnant (D12.5) or non-pregnant female mice. After either 30 min or 24 h of exposure, all major organs were dissected for imaging (n = 7 at each time point) while uterine and mesenteric arteries were dissected for wire myography (n = 6 at each time point). Additional in vitro studies using HMEC-1 endothelial cells were also conducted to investigate the kinetics of interaction between placental nano-vesicles and endothelial cells. Nano-vesicles from first trimester human placentae localized to the lungs, liver and kidneys 24 h after injection into pregnant mice (n = 7). Exposure of pregnant mice to placental nano-vesicles for 30 min in vivo increased the vasodilatory response of mesenteric arteries to acetylcholine, while exposure for 24 h had the opposite effect (P nano-vesicles did not affect the function of uterine arteries or mesenteric arteries from non-pregnant mice. Placental nano-vesicles rapidly interacted with endothelial cells via a combination of phagocytosis, endocytosis and cell surface binding in vitro. N/A. As it is not ethical to administer labelled placental nano-vesicles to

  12. 3D imaging of vesicles in hyaloclastic fragments - clues to syn-eruptive shear conditions

    Science.gov (United States)

    Helo, C.; Flaws, A.; Hess, K.; Franz, A.; Clague, D. A.; Dingwell, D. B.

    2011-12-01

    3D imaging of stretched vesicles in hyaloclastic fragments has been used to investigate the shear environment of mild pyroclastic eruptions at mid-ocean ridges. X-ray computed tomography offers an attractive non-invasive method to investigate geomaterials at a high resolution for the geometry of the different phases. In this study, we have imaged vesicles within two types of basaltic glass fragments. Stretched, ellipsoid-shaped vesicles in thin limu o Pele and tubular vesicles in a pumiceous fragment. Both types originate from pyroclastic activity on Axial Seamount, Juan de Fuca ridge. Rapid quenching of the glass has prevented extensive bubble relaxation and information about syn-eruptive shear and differential stress conditions is stored, as the dimensions of a stretched bubble directly relates to the extent and mode of shearing. The X-ray tomography data was processed using a set of codes based on edge detection and ellipsoid fitting to acquire quantitative information on the shape of the stretched vesicles. Preliminary results demonstrate, that the geometry of the stretched vesicles, e.g., the elongation of the vesicle with respect to the calculated undeformed radius, is in accordance with simple shear scenarios. Stored differential stress ranges from 5 kPa to 90 kPa with shear rates between 3.2x102 s-1 and 5.7x3 s-1 within a single limu o Pele fragment. This range may be explained by either variable time available for relaxation as the cooling front proceeds through the fragment, complex interplay in space and time between fragmentation and quenching, bubble clusters mutually inhibiting each others extend of deformation, or any combination of these. Bubble relaxation time scales are less then 0.005 s providing constraints on the timeframe for cooling to the glass transition. Qualitative analyses of the tube pumice indicates that the tubular structures grow in length by coalescence of vertically aligned ellipsoid-shaped vesicles, and in width by coalescence of

  13. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

    Science.gov (United States)

    Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

    2008-02-15

    Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

  14. Quantal basis of vesicle growth and information content, a unified approach.

    Science.gov (United States)

    Nitzany, Eyal; Hammel, Ilan; Meilijson, Isaac

    2010-09-07

    Secretory vesicles express a periodic multimodal size distribution. The successive modes are integral multiples of the smallest mode (G(1)). The vesicle content ranges from macromolecules (proteins, mucopolysaccharides and hormones) to low molecular weight molecules (neurotransmitters). A steady-state model has been developed to emulate a mechanism for the introduction of vesicles of monomer size, which grow by a unit addition mechanism, G(1)+G(n)-->G(n+1) which, at a later stage are eliminated from the system. We describe a model of growth and elimination transition rates which adequately illustrates the distributions of vesicle population size at steady-state and upon elimination. Consequently, prediction of normal behavior and pathological perturbations is feasible. Careful analysis of spontaneous secretion, as compared to short burst-induced secretion, suggests that the basic character-code for reliable communication should be within a range of only 8-10 vesicles' burst which may serve as a yes/no message. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles

    Science.gov (United States)

    Sharma, S.; LeClaire, M.; Gimzewski, J. K.

    2018-04-01

    Over the last 30 years, atomic force microscopy (AFM) has made several significant contributions to the field of biology and medicine. In this review, we draw our attention to the recent applications and promise of AFM as a high-resolution imaging and force sensing technology for probing subcellular vesicles: exosomes and other extracellular vesicles. Exosomes are naturally occurring nanoparticles found in several body fluids such as blood, saliva, cerebrospinal fluid, amniotic fluid and urine. Exosomes mediate cell-cell communication, transport proteins and genetic content between distant cells, and are now known to play important roles in progression of diseases such as cancers, neurodegenerative disorders and infectious diseases. Because exosomes are smaller than 100 nm (about 30-120 nm), the structural and molecular characterization of these vesicles at the individual level has been challenging. AFM has revealed a new degree of complexity in these nanosized vesicles and generated growing interest as a nanoscale tool for characterizing the abundance, morphology, biomechanics, and biomolecular make-up of exosomes. With the recent interest in exosomes for diagnostic and therapeutic applications, AFM-based characterization promises to contribute towards improved understanding of these particles at the single vesicle and sub-vesicular levels. When coupled with complementary methods like optical super resolution STED and Raman, AFM could further unlock the potential of exosomes as disease biomarkers and as therapeutic agents.

  16. Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)

    Science.gov (United States)

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit S.; Saari, Heikki; Lazaro Ibañez, Elisa; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four "subpopulations" of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.

  17. Intermolecular crosslinks mediate aggregation of phospholipid vesicles by pulmonary surfactant-associated protein SAP-35

    International Nuclear Information System (INIS)

    Ross, G.R.; Sawyer, J.; Whitsett, J.

    1987-01-01

    Pulmonary surfactant-associated protein, Mr=35,000 (SAP-35) is known to bind phospholipids and is hypothesized to function in the organization of surfactant lipid membranes. SAP-35 has been observed to accelerate the calcium-induced aggregation of phospholipid vesicles. In order to define the molecular domains of SAP-35 which function in phospholipid aggregation, they have measured the light scattering properties (400nm) of purified canine SAP-35-phospholipid vesicle suspensions. Accelerated aggregation of unilamellar vesicles, requires SAP-35 and at least 2mM free calcium. The initial rate of A 400 change is proportional to the amount of native SAP-35 added over lipid:protein molar ratios ranging from 100:1 to 5000:1. Removal of the SAP-35 collagen-like domain and a specific cysteine residue involved in intermolecular disulfide bonding by bacterial collagenase digestion destroys the protein's lipid aggregation activity. Pre-incubation of SAP-35 with dithiothreitol (DTT) under nondenaturing conditions also results in a time-dependent loss of aggregation activity. Sucrose density gradient floatation of SAP-35 with 14 C dipalmitoyl phosphatidycholine labelled vesicles in the absence or presence of DTT suggests retention of SAP-35 lipid binding capacity. These data demonstrate the importance of SAP-35 triple helix and disulfide crosslinking integrity for the aggregation of unilamellar phospholipid vesicles

  18. The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

    Directory of Open Access Journals (Sweden)

    Chengliang Zhang

    Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

  19. Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

    International Nuclear Information System (INIS)

    Verdin, Anthony; Lounes-Hadj Sahraoui, Anissa; Newsam, Ray; Robinson, Gary; Durand, Roger

    2005-01-01

    Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities

  20. Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Verdin, Anthony [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France); Lounes-Hadj Sahraoui, Anissa [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)]. E-mail: lounes@univ-littoral.fr; Newsam, Ray [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Robinson, Gary [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Durand, Roger [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)

    2005-01-01

    Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities.

  1. miR-200–containing extracellular vesicles promote breast cancer cell metastasis

    Science.gov (United States)

    Le, Minh T.N.; Hamar, Peter; Guo, Changying; Basar, Emre; Perdigão-Henriques, Ricardo; Balaj, Leonora; Lieberman, Judy

    2014-01-01

    Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200–expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200–dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles. PMID:25401471

  2. Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.

    Science.gov (United States)

    Burrier, R E; Brecher, P

    1983-10-10

    An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.

  3. Onsager's variational principle for the dynamics of a vesicle in a Poiseuille flow

    Science.gov (United States)

    Oya, Yutaka; Kawakatsu, Toshihiro

    2018-03-01

    We propose a systematic formulation of the migration behaviors of a vesicle in a Poiseuille flow based on Onsager's variational principle, which can be used to determine the most stable steady state. Our model is described by a combination of the phase field theory for the vesicle and the hydrodynamics for the flow field. The dynamics is governed by the bending elastic energy and the dissipation functional, the latter being composed of viscous dissipation of the flow field, dissipation of the bending energy of the vesicle, and the friction between the vesicle and the flow field. We performed a series of simulations on 2-dimensional systems by changing the bending elasticity of the membrane and observed 3 types of steady states, i.e., those with slipper shape, bullet shape, and snaking motion, and a quasi-steady state with zig-zag motion. We show that the transitions among these steady states can be quantitatively explained by evaluating the dissipation functional, which is determined by the competition between the friction on the vesicle surface and the viscous dissipation in the bulk flow.

  4. Relationship between volume of the seminal vesicles and sexual activity in middle-aged men.

    Science.gov (United States)

    Taniguchi, H; Kawa, G; Yoshida, K; Takayasu, K; Kinoshita, H; Matsuda, T

    2017-04-01

    The relationship between volume of the seminal vesicles and the frequency of sex and sexual function in middle-aged men is not clear. This study included 81 patients who were diagnosed with localized prostate cancer. Volume of the seminal vesicles was examined using a volume analyser from computed tomography. Sexual function was subjectively evaluated using the Expanded Prostate Cancer Index Composite and Erection Hardness Score. The frequency of sex was surveyed using our original questionnaire. The mean ± SD age of the patients was 67.7 ± 5.3 years. There was no relationship between the volume of seminal vesicles and age of the patients. Volume of the seminal vesicles in patients who answered that they had sexual activity at least once a year was significantly larger than in those who answered no sexual activity for several years (P middle-aged men, volume of the seminal vesicles was significantly larger in those who had a sexual frequency once every 3 months than in those who had a sexual frequency once every 6 months or once a year (P middle-aged men is correlated with sexual activity. © 2016 Blackwell Verlag GmbH.

  5. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  6. Electrogenic Na+-independent Pi transport in canine renal basolateral membrane vesicles

    International Nuclear Information System (INIS)

    Schwab, S.J.; Hammerman, M.R.

    1986-01-01

    To define the mechanism by which Pi exists from the renal proximal tubular cell across the basolateral membrane, we measured 32Pi uptake in basolateral membrane vesicles from dog kidney in the absence of Na+. Preloading of basolateral vesicles with 2 mM Pi transstimulated 32Pi uptake, which is consistent with counterflow. We used measurements of transstimulation to quantitate the transport component of 32Pi uptake. Transstimulation of 32Pi uptake was inhibited less than 30% by concentrations of probenecid as high as 50 mM. In contrast, transstimulation of 35SO4(2-) uptake by intravesicular SO4(2-) was inhibited 92% by 5 mM probenecid. Preloading basolateral vesicles with SO4(2-) did not result in transstimulation of 32Pi uptake. Accumulation of 32Pi in basolateral vesicles above steady state was driven by a membrane potential (intravesicular positive), consistent with Na+-independent Pi transport being accompanied by the net transfer of negative charge across the membrane. We conclude that carrier-mediated, electrogenic Na+-independent 32Pi transport can be demonstrated in basolateral vesicles from dog kidney. This process appears to be mediated, at least in part, via a mechanism different from that by which SO4(2-) is transported. Electrogenic Na+-independent Pi transport may reflect one means by which Pi reabsorbed across the luminal membrane exists from the proximal tubular cell down an electrochemical gradient

  7. The effect of spontaneous curvature on a two-phase vesicle

    International Nuclear Information System (INIS)

    Cox, Geoffrey; Lowengrub, John

    2015-01-01

    Vesicles are membrane-bound structures commonly known for their roles in cellular transport and the shape of a vesicle is determined by its surrounding membrane (lipid bilayer). When the membrane is composed of different lipids, it is natural for the lipids of similar molecular structure to migrate towards one another (via spinodal decomposition), creating a multi-phase vesicle. In this article, we consider a two-phase vesicle model which is driven by nature's propensity to maintain a minimal state of elastic energy. The model assumes a continuum limit, thereby treating the membrane as a closed three-dimensional surface. The main purpose of this study is to reveal the complexity of the Helfrich two-phase vesicle model with non-zero spontaneous curvature and provide further evidence to support the relevance of spontaneous curvature as a modelling parameter. In this paper, we illustrate the complexity of the Helfrich two-phase model by providing multiple examples of undocumented solutions and energy hysteresis. We also investigate the influence of spontaneous curvature on morphological effects and membrane phenomena such as budding and fusion. (paper)

  8. Incorporation of photosenzitizer hypericin into synthetic lipid-based nano-particles for drug delivery and large unilamellar vesicles with different content of cholesterol

    Science.gov (United States)

    Joniova, Jaroslava; Blascakova, Ludmila; Jancura, Daniel; Nadova, Zuzana; Sureau, Franck; Miskovsky, Pavol

    2014-08-01

    Low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are attractive natural occurring vehicles for drug delivery and targeting to cancer tissues. The capacity of both types of the lipoproteins to bind hydrophobic drugs and their functionality as drug carriers have been examined in several studies and it has been also shown that mixing of anticancer drugs with LDL or HDL before administration led to an increase of cytotoxic effects of the drugs in the comparison when the drugs were administered alone. However, a difficult isolation of the lipoproteins in large quantity from a biological organism as well as a variability of the composition and size of these molecules makes practical application of LDL and HDL as drug delivery systems quite complicated. Synthetic LDL and HDL and large unilamellar vesicles (LUV) are potentially suitable candidates to substitute the native lipoproteins for targeted and effective drug delivery. In this work, we have studied process of an association of potent photosensitizer hypericin (Hyp) with synthetic lipid-based nano-particles (sLNP) and large unilamellar vesicles (LUV) containing various amount of cholesterol. Cholesterol is one of the main components of both LDL and HDL particles and its presence in biological membranes is known to be a determining factor for membrane properties. It was found that the behavior of Hyp incorporation into sLNP particles with diameter ca ~ 90 nm is qualitatively very similar to that of Hyp incorporation into LDL (diameter ca. 22 nm) and these particles are able to enter U-87 MG cells by endocytosis. The presence of cholesterol in LUV influences the capacity of these vesicles to incorporate Hyp into their structure.

  9. Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin.

    Directory of Open Access Journals (Sweden)

    Hiruni Harischandra

    2018-04-01

    Full Text Available The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms. Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.

  10. Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

    Science.gov (United States)

    Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

    2017-01-01

    Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

  11. Structure factor of dimyristoylphosphatidylcholine unilamellar vesicles: small-angle x-ray scattering study

    International Nuclear Information System (INIS)

    Kiselev, M.A.; Aksenov, V.L.; Lombardo, D.; Kisselev, A.M.; Lesieur, P.

    2003-01-01

    Small-angle X-ray scattering (SAXS) experiments have been performed on dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles in 40% aqueous sucrose solution. Model of separated form factors was applied for the evaluation of SAXS curves from large unilamellar vesicles. For the first time vesicle structure factor, polydispersity, average radius and membrane thickness were calculated simultaneously from the SAXS curves at T=30 deg C for DMPC concentrations in the range from 15 to 75 mM (1-5% w/w). Structure factor correction to the scattering curve was shown to be negligibly small for the lipid concentration of 15 mM (1% w/w). It was proved to be necessary to introduce structure factor correction to the scattering curves for lipid concentrations ≥ 30 mM (2% w/w)

  12. Munc13 controls the location and efficiency of dense-core vesicle release in neurons.

    Science.gov (United States)

    van de Bospoort, Rhea; Farina, Margherita; Schmitz, Sabine K; de Jong, Arthur; de Wit, Heidi; Verhage, Matthijs; Toonen, Ruud F

    2012-12-10

    Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.

  13. Spontaneous Vesicle Self-Assembly: A Mesoscopic View of Membrane Dynamics

    DEFF Research Database (Denmark)

    Shillcock, J. C.

    2012-01-01

    Amphiphilic vesicles are ubiquitous in living cells and industrially interesting as drug delivery vehicles. Vesicle self-assembly proceeds rapidly from nanometer to micrometer length scales and is too fast to image experimentally but too slow for molecular dynamics simulations. Here, we use...... parallel dissipative particle dynamics (DPD) to follow spontaneous vesicle self-assembly for up to 445 mu s with near-molecular resolution. The mean mass and radius of gyration of growing amphiphilic clusters obey power laws with exponents of 0.85 +/- 0.03 and 0.41 +/- 0.02, respectively. We show that DPD...... provides a computational window onto fluid dynamics on scales unreachable by other explicit-solvent simulations....

  14. Synaptic vesicle exocytosis in hippocampal synaptosomes correlates directly with total mitochondrial volume

    Science.gov (United States)

    Ivannikov, Maxim V.; Sugimori, Mutsuyuki; Llinás, Rodolfo R.

    2012-01-01

    Synaptic plasticity in many regions of the central nervous system leads to the continuous adjustment of synaptic strength, which is essential for learning and memory. In this study, we show by visualizing synaptic vesicle release in mouse hippocampal synaptosomes that presynaptic mitochondria and specifically, their capacities for ATP production are essential determinants of synaptic vesicle exocytosis and its magnitude. Total internal reflection microscopy of FM1-43 loaded hippocampal synaptosomes showed that inhibition of mitochondrial oxidative phosphorylation reduces evoked synaptic release. This reduction was accompanied by a substantial drop in synaptosomal ATP levels. However, cytosolic calcium influx was not affected. Structural characterization of stimulated hippocampal synaptosomes revealed that higher total presynaptic mitochondrial volumes were consistently associated with higher levels of exocytosis. Thus, synaptic vesicle release is linked to the presynaptic ability to regenerate ATP, which itself is a utility of mitochondrial density and activity. PMID:22772899

  15. The basic property of biomembranes: an explanation for the geometric progression of vesicles.

    Science.gov (United States)

    Bont, Willy S

    2009-05-01

    In order to explain the experimentally obtained results with biomembranes it has been assumed, that the diameter of all vesicles, defined by a lipid bilayer, form a perfect crystal. Later on it appeared that biomembranes are liquid crystals. Nevertheless already with biomembranes as crystals it was possible to calculate the diameters of all existing vesicles. As shown in the present paper, the results with liquid crystals were identical to those with a perfect crystal-model. Here I demonstrate that the geometric progression of the diameters of vesicles of biomembranes is based on the entropy of the lipids and it is shown that the diameters of biomembranes fit four geometric series. The surface areas of these series of biomembranes can be approximated by the two geometric series published previously.

  16. Vesicles in Apollo 15 Green Glasses: The Nature of Ancient Lunar Gases

    Science.gov (United States)

    Thomas-Keprta, K. L.; Clemett, S. J.; Berger, E. L.; Rahman, Z.; McKay, D. S.; Gibson, E. K.; Wentworth, S. J.

    2014-01-01

    Detailed studies of Apollo 15 green glass and related beads have shown they were formed in gas-rich fire fountains.. As the magmatic fluid became super-saturated in volatile gas, bubbles or vesicles formed within the magma. These exsolved gases became trapped within vesicles as the glasses were ejected from the fire-fountain and subsequently quenched. One of the keys to understanding formation processes on the ancient moon includes determining the composition of volatile species and elements, including metals, dissolved in magmatic gases. Here we report the nature of mineral phases spatially associated with vesicles in a green glass bead from Apollo sample 15411,42. The phases reflect the composition of the cooling/degassing magmatic vapors and fluids present at the time of bead formation approx, 3 Ga ago

  17. 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.

  18. Small angle neutron scattering and calorimetric studies of large unilamellar vesicles of the phospholipid dipalmitoylphosphatidylcholine

    Energy Technology Data Exchange (ETDEWEB)

    Mason, P.C.; Gaulin, B.D. [Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, L8S 4M1 (CANADA); Epand, R.M. [Department of Biochemistry, McMaster University, Hamilton, Ontario, L8N 3Z5 (CANADA); Wignall, G.D.; Lin, J.S. [Center for Small-Angle Scattering Research, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States)

    1999-03-01

    High-resolution differential scanning calorimetry (DSC) and small angle neutron scattering (SANS) experiments have been conducted on large unilamellar vesicles (LUV{close_quote}s) of the phospholipid dipalmitoylphosphatidylcholine (DPPC) in excess water. The DSC results indicate a phase transition at temperatures corresponding to the gel (L{sub {beta}{sup {prime}}}) to ripple (P{sub {beta}{sup {prime}}}) phase transition seen in multilamellar vesicles of DPPC while the SANS experiments provide direct evidence for the formation of the P{sub {beta}{sup {prime}}} phase in these systems. In addition, it is shown that SANS is an effective technique for extracting structural parameters such as vesicle radius and thickness in LUV model membrane systems. {copyright} {ital 1999} {ital The American Physical Society}

  19. SANS study of the unilamellar DMPC vesicles. The fluctuation model of lipid bilayer

    International Nuclear Information System (INIS)

    Kiselev, M.A.; Zemlyanaya, E.V.; Vinod, A.

    2003-01-01

    On the basis of the separated form-factors model, parameters of the polydispersed unilamellar DMPC vesicle population are analyzed. The neutron scattering length density across the membrane is simulated on the basis of fluctuated model of lipid bilayer. The hydration of vesicle is described by sigmoid distribution function of the water molecules. The results of fitting of the experimental data obtained at the small angle spectrometer SANS-I, PSI (Switzerland) are: average vesicle radius 272±0.4 Armstrong, polydispersity of the radius 27 %, membrane thickness 50.6± Armstrong, thickness of hydrocarbon chain region 21.4±2.8 Armstrong, number of water molecules located per lipid molecule 13±1, and DMPC surface area 59±2 Armstrong 2 . The calculated water distribution function across the bilayer directly explains why lipid membrane is easy penetrated by water molecules

  20. Giant vesicles (GV) in colloidal system under the optical polarization microscope (OPM).

    Science.gov (United States)

    Khalid, Khalisanni; Noh, Muhammad Azri Mohd; Khan, M Niyaz; Ishak, Ruzaina; Penney, Esther; Chowdhury, Zaira Zaman; Hamzah, Mohammad Hafiz; Othman, Maizatulnisa

    2017-09-01

    This paper discusses the unprecedented microscopic findings of micellar growth in colloidal system (CS) of catalyzed piperidinolysis of ionized phenyl salicylate (PS - ). The giant vesicles (GV) was observed under the optical polarization microscope (OPM) at [NaX]=0.1M where X=3-isopropC 6 H 4 O - . The conditions were rationalized from pseudo-first-order rate constant, k obs of PS - of micellar phase at 31.1×10 -3 s -1 reported in previous publication. The overall diameter of GV (57.6μm) in CS (CTABr/NaX/H 2 O)-catalyzed piperidinolysis (where X=3-isopropC 6 H 4 O) of ionized phenyl salicylate were found as giant unilamellar vesicles (GUV) and giant multilamellar vesicles (GMV). The findings were also validated by means of rheological analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Structure Factor of Dimyristoylphosphatidylcholine Unilamellar Vesicles Small-Angle X-Ray Scattering Study

    CERN Document Server

    Kiselev, M A; Kisselev, A M; Lesieur, P; Aksenov, V L

    2003-01-01

    Small-angle X-ray scattering (SAXS) experiments have been performed on dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles in 40 % aqueous sucrose solution. Model of separated form factors was applied for the evaluation of SAXS curves from large unilamellar vesicles. For the first time vesicle structure factor, polydispersity, average radius and membrane thickness were calculated simultaneously from the SAXS curves at T=306{\\circ}C for DMPC concentrations in the range from 15 to 75 mM (1-5 % w/w). Structure factor correction to the scattering curve was shown to be negligibly small for the lipid concentration of 15 mM (1 % w/w). It was proved to be necessary to introduce structure factor correction to the scattering curves for lipid concentrations {\\ge}30 mM (2 % w/w).

  2. Introducing a prognostic score for pretherapeutic assessment of seminal vesicle invasion in patients with clinically localized prostate cancer

    International Nuclear Information System (INIS)

    Salomon, Laurent; Porcher, Raphaeel; Anastasiadis, Aristotelis G.; Levrel, Olivier; Saint, Fabian; Taille, Alexandre de la; Vordos, Dimitrios; Cicco, Antony; Hoznek, Andras; Chopin, Dominique; Abbou, Clement-Claude; Lagrange, Jean-Leon

    2003-01-01

    Purpose: To identify prostate cancer patients who will have the most likely benefit from sparing the seminal vesicles during 3D conformal radiation therapy. Methods and materials: From 1988 to 2001, 532 patients underwent radical prostatectomy for clinically localized prostate cancer. Primary endpoint was the pathological evidence of seminal vesicle invasion. Variables for univariate and multivariate analyses were age, prostate weight, clinical stage, PSA level, Gleason score, number and site of positive prostate sextant biopsies. Multivariate logistic regression with backward stepwise variable selection was used to identify a set of independent predictors of seminal vesicle invasion, and the variable selection procedure was validated by non-parametric bootstrap. Results: Seminal vesicle invasion was reported in 14% of the cases. In univariate analysis, all variables except age and prostate weight were predictors of seminal vesicle invasion. In multivariate analysis, only the number of positive biopsies (P<0.0001), Gleason score (P<0.007) and PSA (P<0.0001) were predictors for seminal vesicles invasion. Based on the multivariate model, we were able to develop a prognostic score for seminal vesicle invasion, which allowed us to discriminate two patient groups: A group with low risk of seminal vesicles invasion (5.7%), and the second with a higher risk of seminal vesicles invasion (32.7%). Conclusions: Using the number of positive biopsies, Gleason score and PSA, it is possible to identify patients with low risk of seminal vesicles invasion. In this population, seminal vesicles might be excluded as a target volume in radiation therapy of prostate cancer

  3. Proton-stimulated Cl-HCO3 antiport by basolateral membrane vesicles of lobster hepatopancreas

    International Nuclear Information System (INIS)

    Ahearn, G.A.; Grover, M.L.; Tsuji, R.T.; Clay, L.P.

    1987-01-01

    Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium 36 Cl uptake experiments at a variety of transmembrane osmotic gradients. 36 Cl uptake into vesicles preloaded with HCO 3 was significantly greater than into vesicles lacking HCO 3 . This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO 3 exchange was potential sensitive and stimulated by an electrically negative vesicle interior. 36 Cl influx (4-s exposures) into HCO 3 -loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and apparent diffusion. 36 Cl influx was a hyperbolic function of both internal [HCO 3 ] and internal [Cl]. The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO 3 being strongly preferred. 36 Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO 3 antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO 3 to power anion exchange and transfer protons against a concentration gradient

  4. A model of Stokesian peristalsis and vesicle transport in a three-dimensional closed cavity.

    Science.gov (United States)

    Aranda, Vivian; Cortez, Ricardo; Fauci, Lisa

    2015-06-25

    The complexity of the mechanics involved in the mammalian reproductive process is evident. Neither an ovum nor an embryo is self-propelled, but move through the oviduct or uterus due to the peristaltic action of the tube walls, imposed pressure gradients, and perhaps ciliary motion. Here we use the method of regularized Stokeslets to model the transport of an ovum or an embryo within a peristaltic tube. We represent the ovum or the embryo as a spherical vesicle of finite volume - not a massless point particle. The outer membrane of the neutrally buoyant vesicle is discretized by nodes that are joined by a network of springs. The elastic moduli of these springs are chosen large enough so that a spherical shape is maintained. For simplicity, here we choose an axisymmetric tube where the geometry of the two-dimensional cross-section along the tube axis reflects that of the sagittal cross-section of the uterine cavity. Although the tube motion is axisymmetric, the presence of the vesicle within the tube requires a fully three-dimensional model. As was found in Yaniv et al. (2009, 2012) for a 2D closed channel, we find that the flow dynamics in a 3D peristaltic tube are strongly influenced by the closed end and the manner in which the peristaltic wave damps out towards the closure. In addition, we demonstrate that the trajectory of a vesicle of finite volume can greatly differ from the trajectory of a massless fluid particle initially placed at the vesicle׳s centroid. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Matrix vesicles in the fibrous cap of atherosclerotic plaque: possible contribution to plaque rupture.

    Science.gov (United States)

    Bobryshev, Y V; Killingsworth, M C; Lord, R S A; Grabs, A J

    2008-10-01

    Plaque rupture is the most common type of plaque complication and leads to acute ischaemic events such as myocardial infarction and stroke. Calcification has been suggested as a possible indicator of plaque instability. Although the role of matrix vesicles in the initial stages of arterial calcification has been recognized, no studies have yet been carried out to examine a possible role of matrix vesicles in plaque destabilization. Tissue specimens selected for the present study represented carotid specimens obtained from patients undergoing carotid endarterectomy. Serial frozen cross-sections of the tissue specimens were cut and mounted on glass slides. The thickness of the fibrous cap (FCT) in each advanced atherosclerotic lesion, containing a well developed lipid/necrotic core, was measured at its narrowest sites in sets of serial sections. According to established criteria, atherosclerotic plaque specimens were histologically subdivided into two groups: vulnerable plaques with thin fibrous caps (FCT <100 microm) and presumably stable plaques, in which fibrous caps were thicker than 100 microm. Twenty-four carotid plaques (12 vulnerable and 12 presumably stable plaques) were collected for the present analysis of matrix vesicles in fibrous caps. In order to provide a sufficient number of representative areas from each plaque, laser capture microdissection (LCM) was carried out. The quantification of matrix vesicles in ultrathin sections of vulnerable and stable plaques revealed that the numbers of matrix vesicles were significantly higher in fibrous caps of vulnerable plaques than those in stable plaques (8.908+0.544 versus 6.208+0.467 matrix vesicles per 1.92 microm2 standard area; P= 0.0002). Electron microscopy combined with X-ray elemental microanalysis showed that some matrix vesicles in atherosclerotic plaques were undergoing calcification and were characterized by a high content of calcium and phosphorus. The percentage of calcified matrix vesicles

  6. Analysing the distribution of synaptic vesicles using a spatial point process model

    DEFF Research Database (Denmark)

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus; Nava, Nicoletta

    2014-01-01

    functionality by statistically modelling the distribution of the synaptic vesicles in two groups of rats: a control group subjected to sham stress and a stressed group subjected to a single acute foot-shock (FS)-stress episode. We hypothesize that the synaptic vesicles have different spatial distributions...... in the two groups. The spatial distributions are modelled using spatial point process models with an inhomogeneous conditional intensity and repulsive pairwise interactions. Our results verify the hypothesis that the two groups have different spatial distributions....

  7. Asymmetrical Polyhedral Configuration of Giant Vesicles Induced by Orderly Array of Encapsulated Colloidal Particles.

    Science.gov (United States)

    Natsume, Yuno; Toyota, Taro

    2016-01-01

    Giant vesicles (GVs) encapsulating colloidal particles by a specific volume fraction show a characteristic configuration under a hypertonic condition. Several flat faces were formed in GV membrane with orderly array of inner particles. GV shape changed from the spherical to the asymmetrical polyhedral configuration. This shape deformation was derived by entropic interaction between inner particles and GV membrane. Because a part of inner particles became to form an ordered phase in the region neighboring the GV membrane, free volume for the other part of particles increased. Giant vesicles encapsulating colloidal particles were useful for the model of "crowding effect" which is the entropic interaction in the cell.

  8. An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents.

    Science.gov (United States)

    Lossouarn, Julien; Dupont, Samuel; Gorlas, Aurore; Mercier, Coraline; Bienvenu, Nadege; Marguet, Evelyne; Forterre, Patrick; Geslin, Claire

    2015-12-01

    Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  9. What can we learn about the lipid vesicle structure from the small angle neutron scattering experiment?

    International Nuclear Information System (INIS)

    Kiselev, M.A.; Zemlyanaya, E.V.; Aswal, V.K.; Neubert, R.H.H.

    2005-01-01

    Small angle neutron scattering (SANS) on the unilamellar vesicle populations (diameter of 500 and 1000 Armstrong) was used to characterize lipid vesicles from dimyristoylphosphatidylcholine (DMPC) in three phases (gel, ripple, and liquid). Parameters of vesicle populations and internal structure of the DMPC bilayer were characterized on the basis of the Separated Form Factor (SFF) model. Vesicle shape changes from about spherical in liquid phase to elliptical in ripple and gel phases for vesicles prepared via extrusion through pores with the diameter of 500 Armstrong. Parameters of the internal bilayer structure (membrane thickness, thickness of the hydrophobic core, hydration, and surface area of lipid molecule) were determined on the basis of the Hydrophobic-Hydrophilic (HH) approximation of neutron scattering length density across the bilayer ρ(x) and on the basis of the Step Function (SF) approximation of ρ(x). It was demonstrated in the framework of HH approximation that DMPC membrane thickness in the liquid phase (T = 30 deg C) depends on the membrane curvature. Vesicle population prepared via extrusion through pores with the diameter of 500 Armstrong is characterized by an average radius of 275.6 ± 0.5 Armstrong, polydispersity of 27%, membrane thickness of 47.8 ± 0.2 Armstrong, thickness of hydrophobic core of 20.5 ± 0.3 Armstrong, surface area per DMPC molecule of 61.0 ± 0.4 A 2 Armstrong, and the number of water molecules per DMPC molecule of 11.9 ± 0.3. Vesicles prepared via extrusion through pores with the diameter of 1000 Armstrong have a polydispersity of 48%, and a membrane thickness of 45.6 ± 0.2 Armstrong. SF approximation was used to describe the DMPC membrane structure in gel (T 10 deg C) and ripple (T = 20 deg C) phases. DMPC vesicles prepared via extrusion through 1000- Armstrong pores have a membrane thickness of 49.6 ± 0.5 Armstrong in the gel phase and 48.3 ± 0.6 Armstrong in the ripple phase. The dependence of the DMPC membrane

  10. Functionalization of Fatty Acid Vesicles through Newly Synthesized Bolaamphiphile-DNA Conjugates

    DEFF Research Database (Denmark)

    Wamberg, M. C.; Wieczorek, R.; Brier, S. B.

    2014-01-01

    The surface functionalization of fatty acid vesicles will allow their use as nanoreactors for complex chemistry. In this report, the tethering of several DNA conjugates to decanoic acid vesicles for molecular recognition and synthetic purposes was explored. Due to the highly dynamic nature......), and consists of a single hydrocarbon chain of 20 carbons having on one end a triazole group linked to the S'-phosphate of the nucleic acid and on the other side a hydroxyl-group. Its insertion was so effective that a fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty...... acid structures....

  11. Extracellular vesicles, tissue factor, cancer and thrombosis – discussion themes of the ISEV 2014 Educational Day

    Directory of Open Access Journals (Sweden)

    Chris Gardiner

    2015-03-01

    Full Text Available Although the association between cancer and venous thromboembolism (VTE has long been known, the mechanisms are poorly understood. Circulating tissue factor–bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV and International Society on Thrombosis and Haemostasis (ISTH held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

  12. Structure-directing star-shaped block copolymers: supramolecular vesicles for the delivery of anticancer drugs.

    Science.gov (United States)

    Yang, Chuan; Liu, Shao Qiong; Venkataraman, Shrinivas; Gao, Shu Jun; Ke, Xiyu; Chia, Xin Tian; Hedrick, James L; Yang, Yi Yan

    2015-06-28

    Amphiphilic polycarbonate/PEG copolymer with a star-like architecture was designed to facilitate a unique supramolecular transformation of micelles to vesicles in aqueous solution for the efficient delivery of anticancer drugs. The star-shaped amphipilic block copolymer was synthesized by initiating the ring-opening polymerization of trimethylene carbonate (TMC) from methyl cholate through a combination of metal-free organo-catalytic living ring-opening polymerization and post-polymerization chain-end derivatization strategies. Subsequently, the self-assembly of the star-like polymer in aqueous solution into nanosized vesicles for anti-cancer drug delivery was studied. DOX was physically encapsulated into vesicles by dialysis and drug loading level was significant (22.5% in weight) for DOX. Importantly, DOX-loaded nanoparticles self-assembled from the star-like copolymer exhibited greater kinetic stability and higher DOX loading capacity than micelles prepared from cholesterol-initiated diblock analogue. The advantageous disparity is believed to be due to the transformation of micelles (diblock copolymer) to vesicles (star-like block copolymer) that possess greater core space for drug loading as well as the ability of such supramolecular structures to encapsulate DOX. DOX-loaded vesicles effectively inhibited the proliferation of 4T1, MDA-MB-231 and BT-474 cells, with IC50 values of 10, 1.5 and 1.0mg/L, respectively. DOX-loaded vesicles injected into 4T1 tumor-bearing mice exhibited enhanced accumulation in tumor tissue due to the enhanced permeation and retention (EPR) effect. Importantly, DOX-loaded vesicles demonstrated greater tumor growth inhibition than free DOX without causing significant body weight loss or cardiotoxicity. The unique ability of the star-like copolymer emanating from the methyl cholate core provided the requisite modification in the block copolymer interfacial curvature to generate vesicles of high loading capacity for DOX with significant

  13. Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Brandauer, Josef; Schjerling, Peter

    2013-01-01

    muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps...... muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP-containing vesicles and protein by 62% (P

  14. Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

    Directory of Open Access Journals (Sweden)

    Ricaurte Lopera-Vásquez

    Full Text Available To evaluate the effect of conditioned media (CM and Extracellular Vesicles (EVs derived from bovine oviduct epithelial cell (BOEC lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E, together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5 EVs/mL, 1.5x10(5 EVs/mL or 7.5x10(4 EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2 and blastocyst development (Day 7-9 was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the

  15. Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma.

    Science.gov (United States)

    Gai, Chiara; Camussi, Francesco; Broccoletti, Roberto; Gambino, Alessio; Cabras, Marco; Molinaro, Luca; Carossa, Stefano; Camussi, Giovanni; Arduino, Paolo G

    2018-04-18

    Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs. OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves. A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC. In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

  16. α-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking.

    Science.gov (United States)

    Lou, Xiaochu; Kim, Jaewook; Hawk, Brenden J; Shin, Yeon-Kyun

    2017-06-06

    Misfolded α-synuclein (A-syn) is widely recognized as the primal cause of neurodegenerative diseases including Parkinson's disease and dementia with Lewy bodies. The normal cellular function of A-syn has, however, been elusive. There is evidence that A-syn plays multiple roles in the exocytotic pathway in the neuron, but the underlying molecular mechanisms are unclear. A-syn has been known to interact with negatively charged phospholipids and with vesicle SNARE protein VAMP2. Using single-vesicle docking/fusion assays, we find that A-syn promotes SNARE-dependent vesicles docking significantly at 2.5 µM. When phosphatidylserine (PS) is removed from t-SNARE-bearing vesicles, the docking enhancement by A-syn disappears and A-syn instead acts as an inhibitor for docking. In contrast, subtraction of PS from the v-SNARE-carrying vesicles enhances vesicle docking even further. Moreover, when we truncate the C-terminal 45 residues of A-syn that participates in interacting with VAMP2, the promotion of vesicle docking is abrogated. Thus, the results suggest that the A-syn's interaction with v-SNARE through its C-terminal tail and its concurrent interaction with PS in trans through its amphipathic N-terminal domain facilitate SNARE complex formation, whereby A-syn aids SNARE-dependent vesicle docking. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.