Membrane Transporters in Citrus clementina Fruit Juice-Derived Nanovesicles

The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D2O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 °C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the protein for arginine and to elucidate structural changes associated with arginine binding. The protein binds arginine with a dissociation constant of 20 mM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor

Minimizing isotropic and deviatoric membrane energy – An unifying formation mechanism of different cellular membrane nanovesicle types

Tiny membrane-enclosed cellular fragments that can mediate interactions between cells and organisms have recently become a subject of increasing attention. In this work the mechanism of formation of cell membrane nanovesicles (CNVs) was studied experimentally and theoretically. CNVs were isolated by centrifugation and washing of blood cells and observed by optical microscopy and scanning electron microscopy. The shape of the biological membrane in the budding process, as observed in phospholipid vesicles, in erythrocytes and in CNVs, was described by an unifying model. Taking the mean curvature h and the curvature deviator d of the membrane surface as the relevant parameters, the shape and the distribution of membrane constituents were determined theoretically by minimization of membrane free energy. Considering these results and previous results on vesiculation of red blood cells it was interpreted that the budding processes may lead to formation of different types of CNVs as regards the compartment (exo/endovesicles), shape (spherical/tubular/torocytic) and composition (enriched/depleted in particular kinds of molecules). It was concluded that the specificity of pinched off nanovesicles derives from the shape of the membrane constituents and not primarily from their chemical identity, which explains evidences on great heterogeneity of isolated extracellular vesicles with respect to composition.</p

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

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 from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addresse

Кераміка «terra sigillata» з с. Зимне на Волині

Стаття присвячена публікації чотирьох керамічних посудин типу «terra sigillata», знайдених на дні р. Луги у с. Зимне Володимир-Волинського району Волинської області. Попередній аналіз цих знахідок дозволяє віднести їх до Понтійського центру виробництва такого посуду. Вірогідним шляхом потрапляння цієї колекції на Волинь була готська експансія у Північне Причорномор’я

Morphology and Formation Mechanisms of Cellular Vesicles Harvested from Blood

Theoretical and experimental evidence on cellular vesicles (CVs) isolated from blood is presented. It is suggested that comparison of the observed shapes with theoretical shapes obtained by minimization of membrane-free energy in combination with electron microscopy is key in the assessment of CV identity. We found that shapes of CVs isolated from blood by repetitive centrifugation (up to 20.000 g) and washing, and observed by scanning electron microscopy (SEM) agreed well with theoretically observed shapes. It is indicated that these CVs are colloids deriving from residual blood cells, mostly platelets. SEM images of washed erythrocytes undergoing budding and transmission electron microscopy (TEM) images of isolated erythrocyte microvesicles likewise showed smooth shapes that we described as characteristic for colloidal CVs. Besides these, the CV isolates may contain other small particles, such as exosomes and viruses, as observed in isolates from tomato homogenate, however, we could not identify such particles in isolates from healthy human blood. Theory of deviatoric elasticity underlaying minimization of the membrane free energy and simulated two-component vesicles with the orientational ordering of anisotropic constituents are presented to indicate the interdependence of curvature—sorting of membrane constituents and their orientational ordering in strongly anisotropically curved regions

Original scientific paper Antimalarial peroxides: the first intramolecular

An intramolecular steroidal 1,2,4,5-tetraoxane has been synthesised in six steps starting from methyl 3-oxo-7�,12�-diacetoxy-5�-cholan-24-oate. The synthesised 1,2,4,5-tetraoxane has moderate in vitro antimalarial activity against P. falciparum strains (IC50 (D6) = 0.35 �g/mL; IC50 (W2) = 0.29 �g/mL)

Isolation of exosome-like vesicles from plants by ultracentrifugation on sucrose/deuterium oxide (D2O) density cushions

Exosomes are nanovesicles of endocytic origin that are about 30-100 nm in diameter, surrounded by a lipid bilayer membrane, and contain proteins, nucleic acids, and other molecules. Mammalian cells- and biological fiuids-derived exosomes have become the subject for a wide range of investigations in biological and biomedical sciences. More recently, a new interest is on the verge of rising: the presence of nanovesicles in plants. Lipoprotein vesicles from apoplastic fiuid and exosome-like vesicles (ELVs) from fruit juice have been isolated and shown that they could be loaded with drugs and uptaken by recipient cells. In order to explore and analyze the contents and functions of ELVs, they must be isolated and purified with intense care. Isolation of ELVs can be a tedious process and often characterized by the co-purification of undesired contaminants. Here we describe a method which isolates ELVs based on their buoyant density. The method utilizes differential centrifugation in step 1 and 1 and 2 M sucrose/deuterium oxide doublecushion ultracentrifugation in step 2, to purify two diverse ELV subpopulations. In this method fruit juice is used as an example of starting material, although this protocol can be used for the isolation of vesicles from apoplastic fiuid too. The quality and the quantity of ELV preparations have been found appropriate for downstream biological and structural studies, like proteomics, transcriptomics, and lipidomics

A new technique in the surgical treatment of Hangman′s fractures: Neurospinal Academy (NSA) technique

Context: Treatment of Hangman′s fractures is still controversial. Hangman′s fractures Type II and IIA are usually treated with surgical procedures. Aim: This study aims at describing the Neurospinal Academy (NSA) technique as an attempt to achieve an approximation of the fracture line to the axis body, which may be used for Type II and IIA patients with severe displacement and angulation. Settings and Design: NSA technique both pars or pedicle screws are placed bicortically to ensure that anterior surface of C2 vertebral body will be crossed 1-2 mm. A rod is prepared in suitable length and curve to connect the two screws. For placing the rod, sufficient amount of bone is resected from the C2 spinous process. C2 vertebral body is pulled back by means of the screws that crossed the anterior surface of C2 vertebral body. Materials and Methods: Hangman II and IIA patient are treated with NSA technique. Result: Angulated and tilted C2 vertebral body was pulled back and approximated to posterior elements. Conclusions: In Hangman′s fractures Type II and IIA with severe vertebral body and pedicle displacement, NSA technique is an effective and reliable treatment alternative for the approximation of posterior elements to the C2 vertebral body, which is tilted, angulated, and dislocated