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      Rapid purification of giant lipid vesicles by microfiltration

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          Abstract

          Giant lipid vesicles (GVs) are emerging models for investigating the properties and reactivity of cell-like microcompartments, providing useful information about plausible protocellular structures in primitive times, as well as for the modern synthetic biology goal of constructing the first artificial cell from its reconstituted and partly modified components. Here we explore a novel methodology of GV purification by microfiltration under reduced pressure, operated by a simple apparatus. The method has been characterized in terms of flow rate, amount of lipid loss, quality of recovered GVs, and size distribution. A case study is reported to show the practicability of GV microfiltration. A clickable fluorescent probe was encapsulated inside GVs; more than 99.9% of the non-entrapped probe was easily and rapidly removed by multiple microfiltrations. This novel methodology is briefly discussed as a future tool for selection experiments on GV populations.

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          Most cited references27

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            A vesicle bioreactor as a step toward an artificial cell assembly.

            An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles containing extracts are produced in an oil-extract emulsion. To form a bilayer the vesicles are transferred into a feeding solution that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk solution expression of enhanced GFP stops after 2 h, inside the vesicle permeability of the membrane to the feeding solution prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the alpha-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein production of 30 muM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst.
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              Giant vesicles: preparations and applications.

              There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
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                Author and article information

                Contributors
                Role: Data curationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: Project administrationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                16 February 2018
                2018
                : 13
                : 2
                : e0192975
                Affiliations
                [1 ] Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Université de Lyon, Claude Bernard Lyon 1, Villeurbanne Cedex, France
                [2 ] Department of Sciences, Roma Tre University, Rome, Italy
                Nagoya University, JAPAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Ecotekne, Lecce, Italy

                Author information
                http://orcid.org/0000-0001-8540-3279
                Article
                PONE-D-17-41631
                10.1371/journal.pone.0192975
                5815610
                29451909
                be07bd9b-dda5-4fbc-a21b-e39d03b48b38
                © 2018 Fayolle et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 November 2017
                : 1 February 2018
                Page count
                Figures: 4, Tables: 2, Pages: 16
                Funding
                Funded by: European COST Action
                Award ID: CM1304
                Funded by: funder-id http://dx.doi.org/10.13039/501100001663, Volkswagen Foundation;
                Award ID: 92850
                Award Recipient :
                This work was carried out and developed within and thanks to the European COST Action CM1304 “Emergence and Evolution of Complex Chemical Systems” (COST-STSM-CM1304-34406). Co-funding from the Volkswagen Foundation (grant no. 92 850) is gratefully acknowledged.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Lipids
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Physical Sciences
                Physics
                Classical Mechanics
                Continuum Mechanics
                Fluid Mechanics
                Fluid Dynamics
                Flow Rate
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Biology and Life Sciences
                Biochemistry
                Lipids
                Lipid Structure
                Physical Sciences
                Materials Science
                Materials by Structure
                Mixtures
                Solutions
                Solutes
                Research and Analysis Methods
                Separation Processes
                Centrifugation
                Physical Sciences
                Chemistry
                Polymer Chemistry
                Macromolecules
                Polymers
                Nylons
                Physical Sciences
                Materials Science
                Materials by Structure
                Polymers
                Nylons
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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                Uncategorized

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