On-chip inverted emulsion method for fast giant vesicle production, handling, and analysis

Liposomes and giant unilamellar vesicles (GUVs) in particular are excellent compartments for constructing artificial cells. Traditionally, their use requires bench-top vesicle growth, followed by experimentation under a microscope. Such steps are time-consuming and can lead to loss of vesicles when they are transferred to an observation chamber. To overcome these issues, we present an integrated microfluidic chip which combines GUV formation, trapping, and multiple separate experiments in the same device. First, we optimized the buffer conditions to maximize both the yield and the subsequent trapping of the vesicles in micro-posts. Captured GUVs were monodisperse with specific size of 18 ± 4 µm in diameter. Next, we introduce a two-layer design with integrated valves which allows fast solution exchange in less than 20 s and on separate sub-populations of the trapped vesicles. We demonstrate that multiple experiments can be performed in a single chip with both membrane transport and permeabilization assays. In conclusion, we have developed a versatile all-in-one microfluidic chip with capabilities to produce and perform multiple experiments on a single batch of vesicles using low sample volumes. We expect this device will be highly advantageous for bottom-up synthetic biology where rapid encapsulation and visualization is required for enzymatic reactions

Dewetting-assisted interface templating : complex emulsions to multicavity particles

Interfacial tension-driven formation of intricate microparticle geometries from complex emulsions is presented in this work. Emulsion-templating is a reliable platform for the generation of a diverse set of microparticles. Here, water-in-styrene-in-water complex emulsions undergo reproducible metamorphosis, i.e., from liquid state emulsions to solid structured microparticles are employed. In contrast to the traditional usage of glass-based microfluidics, polydimethylsiloxane (PDMS) swelling behavior is employed to generate complex emulsions with multiple inner cores. In the presence of block copolymer surfactant, these emulsions undergo gravity-driven dewetting of styrene, to transform into membranous structures with compartments. Further polymerization of styrene skeletal remains resulted in microparticles with interesting geometries and intact membranes. Mechanical and confocal microscopic studies prove the absence of polystyrene within these membranes. Using osmotic pressure, membrane rupture and release of encapsulated gold nanoparticles from such polymerized emulsions leading up to applications in cargo delivery and membrane transport are promoted. Even after membrane rupture, the structured microparticles have shown interesting light-scattering behavior for applications in structural coloring and biosensing. Thereby, proving PDMS-based swelling as a potential methodology for reproducible production of complex emulsions with a potential to be transformed into membranous emulsions or solid microparticles with intricate structures and multiple applications

Rainbows in a bottle : realizing microoptic effects by polymerizable multiple emulsion particle design

In nature, structural colour generation is based on discriminative light propagation associated with physical structures in the range of the wavelengths of light1. These iridescent structural colours are of immense significance2 but not easy to control experimentally and therefore difficult to exploit for applications. In this work, we employ microfluidics to produce polymerizable double emulsions that can not only induce the already known lensing effect3 but also result in the spectral separation of white light. Here, liquids of varying refractive in-dex that constitute the emulsions resulted in patterns of iridescent colours. After polymerization, the inner emulsion cores collapse and this results in curved concave surfaces on these polymeric microspheres. Interestingly, the light propagation along the curved surfaces undergo total internal reflection, followed by near-field interference along exit structures on the polymerized microspheres4. These structured polymeric particles that are able to generate colour dispersions can be exploited for optical devices, displays and even sensing technologies

Cell-free gene expression dynamics in synthetic cell populations

The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment

Specific lipid recruitment by the retroviral gag protein upon HIV-1 assembly : from model membranes to infected cells

The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly

Optimization of the inverted emulsion method for high-yield production of biomimetic giant unilamellar vesicles

In the field of bottom-up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane’s similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion method has great potential for rapid production with high encapsulation of biomolecules. However, the lack of understanding of the parameters that affect production has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize the parameters of the inverted emulsion method to maximize the yield of GUVs. We show that the density difference between the emulsion droplets, oil phase and the outer aqueous phase plays a crucial role in vesicle formation. We investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion volume has on yield and size. Compared to electroformation, our method was not found to alter the membrane mechanics. Finally, we optimize the parameters to minimize the time from workbench to microscope to open up the possibility of time-sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells

Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders

Spatial, temporal, and demographic patterns in prevalence of smoking tobacco use and attributable disease burden in 204 countries and territories, 1990-2019 : a systematic analysis from the Global Burden of Disease Study 2019

Background Ending the global tobacco epidemic is a defining challenge in global health. Timely and comprehensive estimates of the prevalence of smoking tobacco use and attributable disease burden are needed to guide tobacco control efforts nationally and globally. Methods We estimated the prevalence of smoking tobacco use and attributable disease burden for 204 countries and territories, by age and sex, from 1990 to 2019 as part of the Global Burden of Diseases, Injuries, and Risk Factors Study. We modelled multiple smoking-related indicators from 3625 nationally representative surveys. We completed systematic reviews and did Bayesian meta-regressions for 36 causally linked health outcomes to estimate non-linear dose-response risk curves for current and former smokers. We used a direct estimation approach to estimate attributable burden, providing more comprehensive estimates of the health effects of smoking than previously available. Findings Globally in 2019, 1.14 billion (95% uncertainty interval 1.13-1.16) individuals were current smokers, who consumed 7.41 trillion (7.11-7.74) cigarette-equivalents of tobacco in 2019. Although prevalence of smoking had decreased significantly since 1990 among both males (27.5% [26. 5-28.5] reduction) and females (37.7% [35.4-39.9] reduction) aged 15 years and older, population growth has led to a significant increase in the total number of smokers from 0.99 billion (0.98-1.00) in 1990. Globally in 2019, smoking tobacco use accounted for 7.69 million (7.16-8.20) deaths and 200 million (185-214) disability-adjusted life-years, and was the leading risk factor for death among males (20.2% [19.3-21.1] of male deaths). 6.68 million [86.9%] of 7.69 million deaths attributable to smoking tobacco use were among current smokers. Interpretation In the absence of intervention, the annual toll of 7.69 million deaths and 200 million disability-adjusted life-years attributable to smoking will increase over the coming decades. Substantial progress in reducing the prevalence of smoking tobacco use has been observed in countries from all regions and at all stages of development, but a large implementation gap remains for tobacco control. Countries have a dear and urgent opportunity to pass strong, evidence-based policies to accelerate reductions in the prevalence of smoking and reap massive health benefits for their citizens. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd.Peer reviewe