Hybrid organic-inorganic structures trigger the formation of primitive cell-like compartments.

Alkaline hydrothermal vents have become a candidate setting for the origins of life on Earth and beyond. This is due to several key features including the presence of gradients of temperature, redox potential, pH, the availability of inorganic minerals, and the existence of a network of inorganic pore spaces that could have served as primitive compartments. Chemical gardens have long been used as experimental proxies for hydrothermal vents. This paper investigates-10pc]Please note that the spelling of the following author name in the manuscript differs from the spelling provided in the article metadata: Richard J. G. Löffler. The spelling provided in the manuscript has been retained; please confirm. a set of prebiotic interactions between such inorganic structures and fatty alcohols. The integration of a medium-chain fatty alcohol, decanol, within these inorganic minerals, produced a range of emergent 3 dimensions structures at both macroscopic and microscopic scales. Fatty alcohols can be considered plausible prebiotic amphiphiles that might have assisted the formation of protocellular structures such as vesicles. The experiments presented herein show that neither chemical gardens nor decanol alone promote vesicle formation, but chemical gardens grown in the presence of decanol, which is then integrated into inorganic mineral structures, support vesicle formation. These observations suggest that the interaction of fatty alcohols and inorganic mineral structures could have played an important role in the emergence of protocells, yielding support for the evolution of living cells.

Behaviour and the Origin of Organisms.

It is common in origins of life research to view the first stages of life as the passive result of particular environmental conditions. This paper considers the alternative possibility: that the antecedents of life were already actively regulating their environment to maintain the conditions necessary for their own persistence. In support of this proposal, we describe 'viability-based behaviour': a way that simple entities can adaptively regulate their environment in response to their health, and in so doing, increase the likelihood of their survival. Drawing on empirical investigations of simple self-preserving abiological systems, we argue that these viability-based behaviours are simple enough to precede neo-Darwinian evolution. We also explain how their operation can reduce the demanding requirements that mainstream theories place upon the environment(s) in which life emerged.

Quantification of Giant Unilamellar Vesicle Fusion Products by High-Throughput Image Analysis.

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.

Survival strategies of artificial active agents.

Artificial cells can be engineered to display dynamics sharing remarkable features in common with the survival behavior of living organisms. In particular, such active systems can respond to stimuli provided by the environment and undertake specific displacements to remain out of equilibrium, e.g. by moving towards regions with higher fuel concentration. In spite of the intense experimental activity aiming at investigating this fascinating behavior, a rigorous definition and characterization of such "survival strategies" from a statistical physics perspective is still missing. In this work, we take a first step in this direction by adapting and applying to active systems the theoretical framework of Transition Path Theory, which was originally introduced to investigate rare thermally activated transitions in passive systems. We perform experiments on camphor disks navigating Petri dishes and perform simulations in the paradigmatic active Brownian particle model to show how the notions of transition probability density and committor function provide the pivotal concepts to identify survival strategies, improve modeling, and obtain and validate experimentally testable predictions. The definition of survival in these artificial systems paves the way to move beyond simple observation and to formally characterize, design and predict complex life-like behaviors.

Dynamic ordering caused by a source-sink relation between two droplets.

Two droplets composed of different chemicals, 1-decanol and liquid paraffin, floating on the water surface show characteristic co-responsive behavior. The presence of two different types of droplets in the system imposes an asymmetry that would not be possible with single droplets alone. The self-propulsion and interactions between droplets appear because surface active 1-decanol molecules provided by the source are absorbed by the paraffin sink thus generating an asymmetric surface tension gradient. This source-sink relation between droplets stabilizes and enhances the self-propulsion, and leads to a variety of dynamic structures including oscillations in the inter-droplet distance. We found that the character of time evolution also depends on the concentration of dye, Sudan Black B, initially used just to stain the decanol droplet. A simple mathematical model explains the transition between the stationary state and the oscillations as a Hopf bifurcation.

A camphene-camphor-polymer composite material for the production of superhydrophobic absorbent microporous foams.

In a recently published paper (doi.org/10.3390/molecules26113116) on self-propelled motion of objects on the water surface, we described a novel surface-active plastic material obtained by dissolution of camphor and polypropylene in camphene at 250 [Formula: see text]C. The material has wax-like mechanical properties, can be easily formed to any moldable shape, and allows for longer and more stable self-propelled motion if compared with pure camphor or pure camphene or of a camphene-camphor wax. Here we use scanning electron microscopy to visualize and characterize the microporous structure of the solid polypropylene foam formed in the plastic for different polypropylene contents. The topology of foams remaining in the material after camphor and camphene molecules have been removed through evaporation or dissolution is similar to polypropylene foams obtained using thermally-induced phase separation. We show that the foams have a superhydrophobic surface but strongly absorb non-polar liquids, and suggest an array of potential scientific and industrial applications.

A Perfect Plastic Material for Studies on Self-Propelled Motion on the Water Surface.

We describe a novel plastic material composed of camphene, camphor, and polypropylene that seems perfectly suited for studies on self-propelled objects on the water surface. Self-motion is one of the attributes of life, and chemically propelled objects show numerous similarities with animated motion. One of important questions is the relationship between the object shape and its motility. In our previous paper, {R. Löffler et al. PCCP, 2019, 21, 24852-24856}, we presented a novel hybrid material, obtained from the solution of camphor in camphene, that allowed making objects of various shapes. This hybrid material has wax-like mechanical properties, but it has a very high tackiness. Here, we report that a small amount of polypropylene removed this undesirable feature. We investigated the properties of camphor-camphene-polypropylene plastic by performing the statistical analysis of a pill trajectory inside a Petri dish and compared them with those of camphor-camphene wax. The plastic showed the stable character of motion for over an hour-long experiment. The surface activity of objects made of plastic did not significantly depend on the weight ratios of the compounds. Such a significant increase in usefulness came from the polypropylene, which controlled the dissipation of camphor and camphene molecules.

Engineering Life: A Review of Synthetic Biology.

Synthetic biology is a field of scientific research that applies engineering principles to living organisms and living systems. It is a field that is increasing in scope with respect to organisms engineered, practical outcomes, and systems integration. There is a commercial dimension as well, where living organisms are engineered as green technologies that could offer alternatives to industrial standards in the pharmaceutical and petroleum-based chemical industries. This review attempts to provide an introduction to this field as well as a consideration of important contributions that exemplify how synthetic biology may be commensurate or even disproportionate with the complexity of living systems. The engineerability of living systems remains a difficult task, yet advancements are reported at an ever-increasing pace.

Autoselective transport of mammalian cells with a chemotactic droplet.

Liquid chemical droplets, as models of artificial life, when pushed away from equilibrium possess some life-like behaviors such as fission, fusion, movement and chemotaxis. Chemotaxis, directed motion in response to external gradients, is typically an important process in living systems, but certain artificial systems are also capable of this activity. Previously it was shown that droplet-based chemotactic systems when interfaced with biological systems can act as transporters to move cargo such as hydrogel alginate capsules containing living cells. Here the effectiveness of our system to transport different mammalian cell lines (H460, H1299, A549, HEK293T and HS68) was tested. It was discovered that some lung cancer cell lines release surfactants only when placed in the hydrogel capsules. These surfactants establish the interface between the encapsulated cells and the droplet and also support the chemotaxis of the droplet. Because of this, the droplet-mediated transport system is selective for living cells that produce biosurfactants. This is an example of how the integration of artificial life and biological life could be designed where the systems augment each other and function together as a unit. In this case the living system produces the surfactants that the droplet needs for cargo transport and the artificial system provides the transport for the otherwise sessile mammalian cells. Future applications of droplet-based cell handling that is able to distinguish between cells based not only on viability but cell type, developmental stage or other quantifiable traits are considered.

A hybrid camphor-camphene wax material for studies on self-propelled motion.

A new material that combines the self-propelling properties of camphor with the malleability of camphene is reported. It has wax-like mechanical properties at room temperature and can be formed into required shapes. The speed of the self-propelled objects and the trajectory depend on the shape and camphor-camphene weight ratio.

Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels.

Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate starvation. The machinery involves a cascade with a two-component system (TCS) that relays the external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative understanding of this system has implications in synthetic biology and biotechnology, for example, in applications for wastewater treatment. To this aim, we present a computational model and experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together with the mechanisms of gene expression. The model is parameterised within the feasible range, and fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this network that is used in alkaline phosphatase production. Deterministic and stochastic simulations with our model predict the regulation dynamics in higher external phosphate concentrations while reproducing the experimental observations. In a cycle of simulations and experimental verification, our model predicts and explores phenotypes with various synthetic promoter designs that can optimise the inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes have a significant influence over the phosphate response. Together with experimental findings, our model should thus provide insights for the investigations on engineering new sensors and regulators for living technologies.

Regenerated silk fibroin membranes as separators for transparent microbial fuel cells.

In recent years novel applications of bioelectrochemical systems are exemplified by phototrophic biocathodes, biocompatible enzymatic fuel cells and biodegradable microbial fuel cells (MFCs). Herein, transparent silk fibroin membranes (SFM) with various fibroin content (2%, 4% and 8%) were synthesised and employed as separators in MFCs and compared with standard cation exchange membranes (CEM) as a control. The highest real-time power performance of thin-film SFM was reached by 2%-SFM separators: 25.7 ±â€¯7.4 µW, which corresponds to 68% of the performance of the CEM separators (37.7 ±â€¯3.1 µW). Similarly, 2%-SFM revealed the highest coulombic efficiency of 6.65 ±â€¯1.90%, 74% of the CEM efficiency. Current for 2%-SFM reached 0.25 ±â€¯0.03 mA (86% of CEM control). Decrease of power output was observed after 23 days for 8% and 4% and was a consequence of deterioration of SFMs, determined by physical, chemical and biological studies. This is the first time that economical and transparent silk fibroin polymers were successfully employed in MFCs.

Using Imaging Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods.

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 µm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

Vesicle Self-Assembly of Monoalkyl Amphiphiles under the Effects of High Ionic Strength, Extreme pH, and High Temperature Environments.

Vesicles and other bilayered membranous structures can self-assemble from single hydrocarbon chain amphiphiles. Their formation and stability are highly dependent on experimental conditions such as ionic strength, pH, and temperature. The addition of divalent cations, for example, often results in the disruption of vesicles made of a single fatty acid species through amphiphile precipitation. However, membranes composed of amphiphile mixtures have been shown to be more resistant to low millimolar concentrations of divalent cations at room temperature. In this report, several mixtures of amphiphiles are examined for their propensity to self-assemble into membranous vesicular structures under extreme environmental conditions of low pH, high ionic strengths, and temperatures. In particular, mixtures of decylamine with polar cosurfactants were found to efficiently form membranes under these conditions far away from those normally supporting vesicle formation. We further examined decanoic acid/decylamine mixtures in detail. At pH 2 in low ionic strength solutions, the amphiphiles formed oily or crystalline structures; however, the introduction of salts or/and strong acids in conjunction with high temperature induced a stable vesiculation. Thus, extreme environments, such as volcanic or vent environments whose environmental conditions are known to support high chemical reactivity, could have harbored and most significantly promoted the formation of simple organic compartments that preceded cells.

Transport of Live Cells Under Sterile Conditions Using a Chemotactic Droplet.

1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated if such droplets can be used as transporters for living cells. We developed a partially hydrophobic alginate capsule as a protective unit that can be precisely placed in a droplet and transported along chemical gradients. Once the droplets with cargo reached a defined final destination, the association of the alginate capsule and decanol droplet was disrupted and cargo deposited. Both Escherichia coli and Bacillus subtilis cells survived and proliferated after transport even though transport occurred under harsh and sterile conditions.

Multi-Armed Droplets as Shape-Changing Protocells.

Protocells are objects that mimic one or several functions of biological cells and may be embodied as solid particles, lipid vesicles, or droplets. Our work is based on using decanol droplets in an aqueous solution of sodium decanoate in the presence of salt. A decanol droplet under such conditions bears many qualitative similarities with living cells, such as the ability to move chemotactically, divide and fuse, or change its shape. This article focuses on the description of a shape-changing process induced by the evaporation of water from the decanoate solution. Under these conditions, the droplets perform complex shape changes, whereby the originally round decanol droplets grow into branching patterns and mimic the growth of appendages in bacteria or axon growth of neuronal cells. We report two outcomes: (i) the morphological changes are reversible, and (ii) multiple protocells avoid contact between each other during the morphological transformation. The importance of these morphological changes in the context of artificial life are discussed.

Droplets As Liquid Robots.

Liquid droplets are very simple objects present in our everyday life. They are extremely important for many natural phenomena as well as for a broad variety of industrial processes. The conventional research areas in which the droplets are studied include physical chemistry, fluid mechanics, chemical engineering, materials science, and micro- and nanotechnology. Typical studies include phenomena such as condensation and droplet formation, evaporation of droplets, or wetting of surfaces. The present article reviews the recent literature that employs droplets as animated soft matter. It is argued that droplets can be considered as liquid robots possessing some characteristics of living systems, and such properties can be applied to unconventional computing through maze solving or operation in logic gates. In particular, the lifelike properties and behavior of liquid robots, namely (i) movement, (ii) self-division, and (iii) group dynamics, will be discussed.

Primordial membranes: more than simple container boundaries.

Cellular membranes, which are self-assembled bilayer structures mainly composed of lipids, proteins and conjugated polysaccharides, are the defining feature of cell physiology. It is likely that the complexity of contemporary cells was preceded by simpler chemical systems or protocells during the various evolutionary stages that led from inanimate to living matter. It is also likely that primitive membranes played a similar role in protocell 'physiology'. The composition of such ancestral membranes has been proposed as mixtures of single hydrocarbon chain amphiphiles, which are simpler versions of modern lipids. In this review, we discuss the origins, self-assembly patterns, potential functions of these amphiphiles, and their possible roles in protocell activities, as well as their possible evolution towards modern lipids.

Evaporation-Induced Pattern Formation of Decanol Droplets.

Pattern formation in far-from-equilibrium systems is observed in several disciplines including biology, geophysics, and reaction-diffusion chemistry, comprising both living and nonliving systems. We aim to study such nonequilibrium dynamics on the laboratory scale with materials of simple composition. We present a novel system based on a 1-decanol droplet placed in a solution of alkaline decanoate. Previously, we showed the short time scale behavior of this system, which included chemotaxis and maze solving. Here we explore long time scale dynamics of the system (several hours) when open to the environment. We observe dramatic morphological changes in the droplet including long tentacular structures, and we analyze the morphology of these structures at both the macroscopic and microscopic scales across a large range of initial conditions. Such reproducible morphological changes in simple droplets open a path to the exploration of shape-based effects in larger-scale pattern-formation studies.

Specific and Reversible DNA-Directed Self-Assembly of Modular Vesicle-Droplet Hybrid Materials.

Modular hybrid structures functionalized to assemble in a controlled manner possess diverse properties necessary for a new generation of complex materials and applications. Here, we functionalized giant unilamellar vesicles and emulsion droplets with biotinylated single-stranded DNA oligonucleotides using streptavidin as an intermediary linker to demonstrate specific and reversible DNA-directed self-assembly into vesicle-droplet hybrid structures. A low molar percentage of PEGylated phospholipids independent of the DNA-based recognition machinery at the supramolecular surface modulated the stability of the system. The reversibility of the aggregation was demonstrated by heating the hybrid structures above the melting temperature of the conjoining double-stranded DNA in the presence of excess biotin. The application of this general assembly control system to diverse multiphase soft materials provides the mechanism to assemble complex modular hybrid systems in a controllable and reversible way, which may provide an advantage where multifunctionality is a target property.