Low-cost equipment for electroformation of Giant Unilamellar Vesicles

Bellon, Javier Alejandro; Pino, Marcelo Javier; Wilke, Natalia

doi:10.1016/j.ohx.2018.e00037

Cited by 16 publications

(12 citation statements)

References 13 publications

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“…In contrast to "classical" liposomes, GUVs are much larger in size (1 -100 µm) and can directly be observed by light microscopic techniques. 168 On the downside, GUVs are less robust and their preparation is less straightforward (see [169][170][171][172][173][174][175][176][177][178] for literature on GUV formation). Compared to SUVs, GUVs have a vastly increased surface and inner volume that allows for the encapsulation of entire protein machineries, small vesicles or even whole bacteria, in order to mimic increasingly complex functions of living cells.…”

Section: Towards a Synthetic Cellmentioning

confidence: 99%

Current problems and future avenues in proteoliposome research

Amati

Graf

Deutschmann

et al. 2020

Biochemical Society Transactions

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Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

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Section: Towards a Synthetic Cellmentioning

confidence: 99%

Current problems and future avenues in proteoliposome research

Amati

Graf

Deutschmann

et al. 2020

Biochemical Society Transactions

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“…For example, in an industrial or quasi-industrial granulator [70] used to make flakes or chips, the produced material can be converted to low-quality filament, which can then be subsequently chopped by the invention discussed here and then converted to high-quality 3-D printing filament. This filament can be tuned for specific properties such as those needed for scientific hardware [71][72][73][74]. This becomes important as manufacturers begin to disclose the materials from which they are made [75] in order to facilitate recycling and/or market opportunities from consumers understanding the material ingredients that make up their products.…”

Section: Fractional Control Of Color and Composite Mixingmentioning

confidence: 99%

3-D Printable Polymer Pelletizer Chopper for Fused Granular Fabrication-Based Additive Manufacturing

Woern

Pearce

2018

Inventions

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Although distributed additive manufacturing can provide high returns on investment, the current markup on commercial filament over base polymers limits deployment. These cost barriers can be surmounted by eliminating the entire process of fusing filament by three-dimensional (3-D) printing products directly from polymer granules. Fused granular fabrication (FGF) (or fused particle fabrication (FPF)) is being held back in part by the accessibility of low-cost pelletizers and choppers. An open-source 3-D printable invention disclosed here allows for precisely controlled pelletizing of both single thermopolymers as well as composites for 3-D printing. The system is designed, built, and tested for its ability to provide high-tolerance thermopolymer pellets with a number of sizes capable of being used in an FGF printer. In addition, the chopping pelletizer is tested for its ability to chop multi-materials simultaneously for color mixing and composite fabrication as well as precise fractional measuring back to filament. The US$185 open-source 3-D printable pelletizer chopper system was successfully fabricated and has a 0.5 kg/h throughput with one motor, and 1.0 kg/h throughput with two motors using only 0.24 kWh/kg during the chopping process. Pellets were successfully printed directly via FGF as well as indirectly after being converted into high-tolerance filament in a recyclebot.

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“…GUVs were prepared by the electroformation technique as described by Angelova and Dimitrov (Angelova and Dimitrov, 1986) using a home-made wave generator and a chamber with stainless steel electrodes (Bellon et al, 2018). Briefly, 7 µ L of a 0.5 mM lipid solution (in chloroform) were spread on two stainless steel electrodes, and were left under vacuum for at least 3h to remove all traces of the organic solvent.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the activity of an antimicrobial peptide by sterols or hopanoids: possible role in cell recognition

Alvares

Monti

Neto

et al. 2020

Preprint

Self Cite

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It is now accepted that hopanoids act as sterol-surrogates in membranes of some sterol-lacking bacteria. Here 10 we inquiry whether the hopanoid diplopterol (DP) could attenuate the activity of the antimicrobial peptide Polybia-MP1 11 (MP1) similarly to cholesterol (CHO). Survival of P. aeruginosa exposed to MP1 was lower for cells incubated with DP than 12 those incubated with CHO, and the affinity and subsequent effect of the peptide on lipid bilayers were different in the 13 presence of DP than in the presence of CHO. Membrane properties showed a non-monotonic behavior as the peptide 14 adsorbed, penetrated, and translocated bilayers with DP suggesting a reorganization of MP1 during these processes. We 15 conclude that MP1 selectivity is finely tuned by lipid composition, and propose the differential interaction and consequent 16 effect promoted by the peptide in membranes with diplopterol as a promising starting point for targeting antimicrobial 17 peptides to hopanoid-containing bacterial membranes. 18Introduction 19 Cell membranes are simultaneously compact and fluid. These properties, which are conserved in different species, have 20 been proposed to be those that are compatible with life (Heimburg, 2007), and it is accepted that sterols are inducers 21 of these particular mechanical conditions in different kingdoms. It has been shown in pioneer works and recent publi-22 cations that hopanoids can be considered sterol-surrogates in sterol-lacking bacteria. Biophysical studies, using artificial 23 membranes, have demonstrated that hopanoids decrease the membrane permeability and increase the membrane or-24 der without compromising their mechanical properties (Sáenz et al., 2012(Sáenz et al., , 2015 Mangiarotti et al., 2019a). In addition, 25 and in accordance with in vitro studies, hopanoids promote resistance to antibiotics, detergents, extreme pHs, high tem-26 perature, oxidation, and high osmolarity in bacteria (Sáenz, 2010; Belin et al., 2018; Welander et al., 2010; Malott et al., 27 2014; Schmerk et al., 2011; Silipo et al., 2014; Kulkarni et al., 2013). 28 Given the similarities between membranes with hopanoids and those with sterols, here we compared the activity of a 29 known antimicrobial peptide (AMP), Polybia-MP1 (MP1, IDWKKLLDAAKQIL-NH 2 ), against membranes with diplopterol (DP, 30 a major hopanoid) or cholesterol (CHO). This peptide, extracted from the venom of the Brazilian wasp Polybia paulista, 31 exhibits bactericidal activity against Gram-positive and Gram-negative bacteria being non-hemolytic and non-cytotoxic 32 (Souza et al., 2005). The aim of this study is to get an insight into the reasons for this peptide behavior, deepening the 33 understanding of the dependence of membrane composition on the MP1-membrane interaction. We inquired whether 34 diplopterol protects membranes from peptide action as good as cholesterol, which hinders membrane disruption by 35 MP1 (Dos Santos Cabrera et al., 2008, 2012. We showed using in vivo assays that the presence of DP prevents the a...

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Low-cost equipment for electroformation of Giant Unilamellar Vesicles

Cited by 16 publications

References 13 publications

Current problems and future avenues in proteoliposome research

Current problems and future avenues in proteoliposome research

3-D Printable Polymer Pelletizer Chopper for Fused Granular Fabrication-Based Additive Manufacturing

Regulation of the activity of an antimicrobial peptide by sterols or hopanoids: possible role in cell recognition

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