Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device.

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides.

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1-16 mol % smooth LPS (corresponding to 16-79 wt %), while vesicles with inner leaflet LPS reach coverages of 2-7 mol % smooth LPS (28-60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Discrete Hamstring: Quadriceps Strength Ratios Do Not Represent Angle-Specific Ratios in Premier League Soccer Players.

ABSTRACT: Lunn, DE, Nicholson, G, Cooke, M, Crespo, R, Robinson, T, Price, RJ, and Walker, J. Discrete hamstring: quadriceps strength ratios do not represent angle-specific ratios in Premier League soccer players. J Strength Cond Res 37(12): 2417-2422, 2023-This study compared angle-specific hamstring:quadriceps (H:Q) ratios with their discrete counterparts during strength testing in professional male soccer players. Twenty-seven professional English Premier League soccer players were recruited for this study (age: 22 ± 4 years; stature: 1.81 ± 0.08 m; body mass: 74.7 ± 6.5 kg). Isokinetic testing of the knee flexors and extensors was conducted concentrically at two angular velocities (60° and 240°·s -1 ) and eccentrically (for the knee flexors only) at 30°·s -1 . Conventional H:Q ratio was calculated as the ratio between peak joint moment in the flexors and extensors at 60°·s -1 . Functional H:Q ratio was calculated as the peak joint moment in the flexors during the eccentric condition and the extensors at 240°·s -1 . Discrete conventional and functional H:Q ratios were 0.56 ± 0.06 and 1.28 ± 0.22, respectively. The residual differences between discrete values and angle-specific residual values were 13.60 ± 6.56% when normalized to the magnitude of the discrete value. For the functional ratios, the normalized residual was 21.72 ± 5.61%. Therefore, neither discrete ratio was representative of angle-specific ratios, although the conventional ratio had lower error overall. Therefore, practitioners should consider H:Q ratio throughout the full isokinetic range of motion, not just the discrete ratio calculated from peak joint moments, when designing and implementing training programs or monitoring injury risk, recovery from injury, and readiness to return to play.

Opportunities arising from the COVID-19: an international orthopaedic surgeons' perspective.

PURPOSE: The unprecedented COVID-19 experience has posed severe challenges to the health care system and several of these are documented in orthopaedic surgery; however, although the pandemic has also brought positive changes, these have not been precisely documented. The purpose of this survey is to identify positive perceptions by orthopaedic surgeons at an international level. METHODS: A cross-sectional, web-based survey inviting 120 orthopaedic surgeons was conducted in April 2020 querying about the positive lessons COVID-19 would teach us. From all responses, thematic codes were obtained and an exploratory thematic analysis was carried out to determine the prevalent themes. RESULTS: A total of 100 responses (83% response rate) from a total of seven countries were received. The variety of responses received were grouped into 13 different thematic codes. The thematic analysis generated two major themes: "Virtual reorganization" and "Wellness and sustainability". Fifty-four per cent of the participants reported positive changes in service reorganization and virtual consultation, whereas 30% replied with an increased feeling of well-being which overlapped with environmental benefits, including reduced paperwork, reduced travelling and increased quality time for family and reflection. CONCLUSIONS: Despite the negative aspects of the pandemic, responders reported several positive changes particularly relating to service reorganization and personal well-being. This study prompts further larger scale research to unravel further detail in those positive aspects and strongly enhance our future orthopaedic practice.

Use of scenario ensembles for deriving seismic risk.

High death tolls from recent earthquakes show that seismic risk remains high globally. While there has been much focus on seismic hazard, large uncertainties associated with exposure and vulnerability have led to more limited analyses of the potential impacts of future earthquakes. We argue that as both exposure and vulnerability are reducible factors of risk, assessing their importance and variability allows for prioritization of the most effective disaster risk-reduction (DRR) actions. We address this through earthquake ensemble modeling, using the example of Nepal. We model fatalities from 90 different scenario earthquakes and establish whether impacts are specific to certain scenario earthquakes or occur irrespective of the scenario. Our results show that for most districts in Nepal impacts are not specific to the particular characteristics of a single earthquake, and that total modeled impacts are skewed toward the minimum estimate. These results suggest that planning for the worst-case scenario in Nepal may place an unnecessarily large burden on the limited resources available for DRR. We also show that the most at-risk districts are predominantly in rural western Nepal, with ∼9.5 million Nepalis inhabiting districts with higher seismic risk than Kathmandu. Our proposed approach provides a holistic consideration of seismic risk for informing contingency planning and allows the relative importance of the reducible components of risk (exposure and vulnerability) to be estimated, highlighting factors that can be targeted most effectively. We propose this approach for informing contingency planning, especially in locations where information on the likelihood of future earthquakes is inadequate.

Graphitic Carbon Nitride Stabilizers Meet Microfluidics: From Stable Emulsions to Photoinduced Synthesis of Hollow Polymer Spheres.

Graphitic carbon nitride (g-CN) has been utilized as a heterogeneous catalyst, but is usually not very well dispersible. The amphiphilic character of g-CN can be altered by surface modifications of g-CN nanopowders. Introducing hydrophilicity or hydrophobicity is a promising avenue for producing advanced emulsion systems. In this study, a special surface-modified g-CN is used to form stable Pickering emulsions. Using a PDMS-based microfluidic device designed for stable production of both single and double emulsions, it is shown that surface-modified g-CNs allow the manufacture of unconventionally stable and precise Pickering emulsions. Shell thickness of the double emulsions is varied to emphasize the robustness of the device and also to demonstrate the extraordinary stabilization brought by the surface-modified carbon nitride used in this study. Due to the electrostatic stabilization also in the oil phase, double emulsions are centered. Finally, when produced from polymerizable styrene, hollow polymer microparticles are formed with precise and tunable sizes, where g-CN is utilized as the only stabilizer and photoinitiator.

Precipitation of Calcium Carbonate Inside Giant Unilamellar Vesicles Composed of Fluid-Phase Lipids.

Biomineralization of CaCO3 commonly involves the formation of amorphous CaCO3 precursor particles that are produced in a confined space surrounded by a lipid bilayer. While the influence of confinement itself has been investigated with different model systems, the impact of an enclosing continuous lipid bilayer on CaCO3 formation in a confined space is still poorly understood as appropriate model systems are rare. Here, we present a new versatile method based on droplet-based microfluidics to produce fluid-phase giant unilamellar vesicles (GUVs) in the presence of high CaCl2 concentrations. These GUVs can be readily investigated by means of confocal laser scanning microscopy in combination with bright-field microscopy, demonstrating that the formed CaCO3 particles are in conformal contact with the fluid-phase lipid bilayer and thus suggesting a strong interaction between the particle and the membrane. Atomic force microscopy adhesion studies with membrane-coated spheres on different CaCO3 crystals corroborated this notion of a strong interaction between the lipids and CaCO3.

Membrane permeability to water measured by microfluidic trapping of giant vesicles.

We use a microfluidic method to estimate the water permeability coefficient (p) of membranes. As model lipid membranes we employ giant unilamellar vesicles (GUVs) composed of palmitoyloleoyl phosphatidylcholine and cholesterol (10 mol%). We have developed a microfluidic device with multiple chambers to trap GUVs and allow controlled osmotic exchange. Each chamber has a ring-shaped pressure-controlled valve which upon closure allows isolation of the GUVs in a defined volume. Opening the valves leads to a rapid fluid exchange between the trapping region and the microchannel network outside, thus allowing precise control over solution concentration around the GUVs contrary to other experimental approaches for permeability measurements reported in the literature. The area and volume changes of individual vesicles are monitored with confocal microscopy. The solute concentration in the immediate vicinity of the GUVs, and thus the concentration gradient across the membrane, is independently assessed. The data are well fitted by a simple model for water permeability which assumes that the rate of change in volume of a GUV per unit area is linearly proportional to concentration difference with permeability as the proportionality constant. Experiments of GUV osmotic deflation with hypertonic solutions yield the permeability of POPC/cholesterol 9/1 membranes to be p = 15.7 ± 5.5 µm s-1. For comparison, we also show results using two other approaches, which either do not take into account local concentration changes and/or do not resolve the precise vesicle shape. We point out the errors associated with these limitations. Finally, we also demonstrate the applicability of the microfluidic device for studying the dynamics of vesicles under flow.

Altered thyroid hormone levels affect the capacity for temperature-induced developmental plasticity in larvae of Rana temporaria and Xenopus laevis.

Anuran larvae show phenotypic plasticity in age and size at metamorphosis as a response to temperature variation. The capacity for temperature-induced developmental plasticity is determined by the thermal adaptation of a population. Multiple factors such as physiological responses to changing environmental conditions, however, might influence this capacity as well. In anuran larvae, thyroid hormone (TH) levels control growth and developmental rate and changes in TH status are a well-known stress response to sub-optimal environmental conditions. We investigated how chemically altered TH levels affect the capacity to exhibit temperature-induced developmental plasticity in larvae of the African clawed frog (Xenopus laevis) and the common frog (Rana temporaria). In both species, TH level influenced growth and developmental rate and modified the capacity for temperature-induced developmental plasticity. High TH levels reduced thermal sensitivity of metamorphic traits up to 57% (R. temporaria) and 36% (X. laevis). Rates of growth and development were more plastic in response to temperature in X. laevis (+30%) than in R. temporaria (+6%). Plasticity in rates of growth and development is beneficial to larvae in heterogeneous habitats as it allows a more rapid transition into the juvenile stage where rates of mortality are lower. Therefore, environmental stressors that increase endogenous TH levels and reduce temperature-dependent plasticity may increase risks and the vulnerability of anuran larvae. As TH status also influences metabolism, future studies should investigate whether reductions in physiological plasticity also increases the vulnerability of tadpoles to global change.

Reversible pH-Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions.

In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pKa , to drive liquid-liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pKa . We further show that pH-triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi-compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom-up synthetic biology approach.

Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study.

Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs, large unilamellar vesicles) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine (POPC)/palmitoyloleoylphosphatidylglycerol (POPG) molar ratios. By using a set of optical-microscopy- and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%) in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charge-dependent manner; the vesicle area increase is deduced from GUV electrodeformation. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs using fluorescence resonance energy transfer (FRET), which leads to consistent results when compared to fluorescence-lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. The results suggest that fusion is driven by membrane charge and appears to proceed up to charge neutralization of the acceptor GUV.

Observations of Membrane Domain Reorganization in Mechanically Compressed Artificial Cells.

Giant unilamellar vesicles (GUVs) are considered to be the gold standard for assembling artificial cells from the bottom up. In this study, we investigated the behavior of such biomimetic vesicles as they were subjected to mechanical compression. A microfluidic device is presented that comprises a trap to capture GUVs and a microstamp that is deflected downwards to mechanically compress the trapped vesicle. After characterization of the device, we show that single-phase GUVs can be controllably compressed to a high degree of deformation (D=0.40) depending on the pressure applied to the microstamp. A permeation assay was implemented to show that vesicle bursting is prevented by water efflux. Next, we mechanically compressed GUVs with co-existing liquid-ordered and liquid-disordered membrane phases. Upon compression, we observed that the normally stable lipid domains reorganized themselves across the surface and fused into larger domains. This phenomenon, observed here in a model membrane system, not only gives us insights into how the multicomponent membranes of artificial cells behave, but might also have interesting consequences for the role of lipid rafts in biological cells that are subjected to compressive forces in a natural environment.

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-based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion-based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way 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.

Special Issue on Bottom-Up Synthetic Biology.

Bottom-up synthetic biology uses both biological and artificial chemical building blocks to create biomimetic systems, including artificial cells. Existing and new technologies such as microfluidics are being developed and applied to the assembly processes. In this special issue, experts present and review the latest progress in this rapidly expanding and diverse field.

MaxSynBio: Avenues Towards Creating Cells from the Bottom Up.

A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

Solution Asymmetry and Salt Expand Fluid-Fluid Coexistence Regions of Charged Membranes.

Liquid-liquid phase separation in giant unilamellar vesicles (GUVs) leads to the formation of intramembrane domains. To mimic charged biological membranes, we studied phase separation and domain formation in GUVs of ternary lipid mixtures composed of egg sphingomyelin, cholesterol, and the negatively charged lipid dioleoylphosphatidylglycerol. The GUVs were exposed to solutions of sucrose and high-saline buffer. The phase diagram was determined using epifluorescence microscopy for vesicle populations with symmetric and asymmetric solution compositions across the membranes. Trans-membrane solution asymmetry was found to affect the membrane phase state. Furthermore, compared to the case of salt-free conditions, the phase diagram in the presence of high-saline buffer (both symmetrically or asymmetrically present across the membrane) was found to exhibit a significantly extended region of liquid-ordered and liquid-disordered coexistence. These observations were confirmed on single GUVs using microfluidics and confocal microscopy. Moreover, we found that the miscibility temperatures markedly increased for vesicles in the presence of symmetric and asymmetric salt solutions. Our results demonstrate a substantial effect of salt and solution asymmetry on the phase behavior of charged membranes, which has direct implications for protein adsorption onto these membranes and for the repartitioning of proteins within the membrane domains.

Membranes under shear stress: visualization of non-equilibrium domain patterns and domain fusion in a microfluidic device.

In this study we investigate the effect of shear force on lipid membranes induced by external fluid flow. We use giant unilamellar vesicles (GUVs) as simple cell models and chose a ternary lipid mixture that exhibits liquid-ordered and liquid-disordered domains. These domains are stained with different dyes to allow visualization of changes within the membrane after the application of flow. A microfluidic device served as a valuable platform to immobilize the vesicles and apply shear forces of a defined strength. Moreover, integration of valves allowed us to stop the flow instantaneously and visualize the relaxing domain patterns by means of high-resolution fluorescence microscopy. We observed the formation of transient, non-deterministic patterns of the formerly round domains during application of flow. When the flow is stopped, round domains are formed again on a time scale of ms to s. At longer time scales of several seconds to minutes, the domains fuse into larger domains until they reach equilibrium. These processes are accelerated with increasing temperature and vesicles with budding domains do not fuse unless the temperature is elevated. Our results show the strong effect of the flow on the lipid membrane and we believe that this phenomenon plays a crucial role in the processes of mechanotransduction in living cells.

The psychopathology of James Bond and its implications for the revision of the DSM-(00)7.

OBJECTIVE: To develop a more concise, user-friendly edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM). The DSM advisory board is probably already hard at work on the DSM-6, so this study is focused on the DSM-(00)7 edition. DESIGN: We conducted an observational study, using a mixed methods approach to analyse the 50th edition boxset of James Bond experiences. James Bond was selected as a suitably complex subject for the basis of a trial of simplifying the DSM. SETTING: Researchers' televisions and computers from late January to mid-April in Auckland, New Zealand. RESULTS: Following a review of the 23 James Bond video observations, we identified 32 extreme behaviours exhibited by the subject; these could be aggregated into 13 key domains. A Delphi process identified a cluster of eight behaviours that comprise the Bond Adequacy Disorder (BAD). A novel screening scale was then developed, the Bond Additive Descriptors of Anti-Sociality Scale (BADASS), with a binary diagnostic outcome, BAD v Normality Disorder. We propose that these new diagnoses be adopted as the foundation of the DSM-(00)7. CONCLUSIONS: The proposed DSM-(00)7 has benefits for both patients and clinicians. Patients will experience reduced stigma, as most individuals will meet the criteria for Normality Disorder. This parsimonious diagnostic approach will also mean clinicians have more time to focus on patient management.

Interaction of ß(3) /ß(2) -peptides, consisting of Val-Ala-Leu segments, with POPC giant unilamellar vesicles (GUVs) and white blood cancer cells (U937)--a new type of cell-penetrating peptides, and a surprising chain-length dependence of their vesicle- and cell-lysing activity.

Many years ago, ß(2) /ß(3) -peptides, consisting of alternatively arranged ß(2) - and ß(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All ß(3) /ß(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric ß(3) /ß(2) -, ß(3) -, and ß(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of ß(3) - or ß(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the ß(3) /ß(2) -nonamer and/or the ß(3) /ß(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the ß(3) /ß(2) -nona- and ß(3) /ß(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' ß(3) /ß(2) -peptides with membranes and to evaluate possible biomedical applications.