Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

Droplet-Microarray: Miniaturized Platform for High-Throughput Screening of Antimicrobial Compounds.

Currently, there are no time-saving and cost-effective high-throughput screening methods for the evaluation of bacterial drug-resistance. In this study, a droplet microarray (DMA) system is established as a miniaturized platform for high-throughput screening of antibacterial compounds using the emerging, opportunistic human pathogen Pseudomonas aeruginosa (P. aeruginosa) as a target. Based on the differences in wettability of DMA slides, a rapid method for generating microarrays of nanoliter-sized droplets containing bacteria is developed. The bacterial growth in droplets is evaluated using fluorescence. The new method enables immediate screening with libraries of antibiotics. A novel simple colorimetric readout method compatible with the nanoliter size of the droplets is established. Furthermore, the drug-resistance of P. aeruginosa 49, a multi-resistant strain from an environmental isolate, is investigated. This study demonstrates the potential of the DMA platform for the rapid formation of microarrays of bacteria for high-throughput drug screening.

Topographic cues guide the attachment of diatom cells and algal zoospores.

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.

Collaborative Action of Surface Chemistry and Topography in the Regulation of Mesenchymal and Epithelial Markers and the Shape of Cancer Cells.

Malignant transformation is associated with enhancement of cell plasticity, which allows cancer cells to survive under different conditions by adapting to their microenvironment during growth and metastatic spread. Much effort has been devoted to understanding the molecular mechanisms of these processes. Although the importance of the extracellular matrix and of surface properties in these mechanisms is evident, the direct impact of distinct physical and chemical surfaces characteristics on cell fate remains unclear. Here, we have addressed this question using HT1080 fibrosarcoma cells as a model. To examine the relationship between surface topography, chemistry, and cell behavior, hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA-EDMA) and hydrophilic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) surfaces with three different topographies (microporous, nanoporous, and nonporous) were generated. These surfaces were then modified by photoinitiated grafting of three different methacrylate monomers to create surface chemistry gradients of either negatively (AMPS) or positively (META) charged or zwitterionic (MDSA) functionalities. Our results show that AMPS promotes cell spreading, but that META abolishes cell growth. META and MDSA grafted on microporous BMA-EDMA produced superhydrophilic surfaces with high globularity and elasticity, which modified the cell phenotype by inhibiting cell spreading, followed by loss of mesenchymal characteristics and a reduction in protein levels of the mesenchymal markers N-cadherin, beta-catenin, p120 catenin, and also of the adaptor proteins vinculin and paxillin that are associated with adhesion and cancer cell invasion. The effect was strengthened along the gradient, suggesting that the density of the functional groups plays a role in this process. On the nanoporous surface, only MDSA grafting resulted in a significant increase in cell number, a reduction in N-cadherin expression, increased beta-catenin and p120 catenin levels, as well as the appearance of the epithelial marker E-cadherin. This indicates that the cancer cells have a high plasticity that is triggered by the collaborative effect of physical and chemical surface properties.

Shear-Induced Detachment of Polystyrene Beads from SAM-Coated Surfaces.

In this work we experimentally and theoretically analyze the detachment of microscopic polystyrene beads from different self-assembled monolayer (SAM) surfaces in a shear flow in order to develop a mechanistic model for the removal of cells from surfaces. The detachment of the beads from the surface is treated as a thermally activated process applying an Arrhenius Ansatz to determine the activation barrier and attempt frequency of the rate determing step in bead removal. The statistical analysis of the experimental shear detachment data obtained in phosphate-buffered saline buffer results in an activation energy around 20 kJ/mol, which is orders of magnitude lower than the adhesion energy measured by atomic force microscopy (AFM). The same order of magnitude for the adhesion energy measured by AFM is derived from ab initio calculations of the van der Waals interaction energy between the polystyrene beads and the SAM-covered gold surface. We conclude that the rate determing step for detachment of the beads is the initiation of rolling on the surface (overcoming static friction) and not physical detachment, i.e., lifting the particle off the surface.

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces.

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a 'normal' manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo 'pseudosettlement' whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a 'hit and stick' motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.

Reactive superhydrophobic surface and its photoinduced disulfide-ene and thiol-ene (bio)functionalization.

Reactive superhydrophobic surfaces are highly promising for biotechnological, analytical, sensor, or diagnostic applications but are difficult to realize due to their chemical inertness. In this communication, we report on a photoactive, inscribable, nonwettable, and transparent surface (PAINTS), prepared by polycondensation of trichlorovinylsilane to form thin transparent reactive porous nanofilament on a solid substrate. The PAINTS shows superhydrophobicity and can be conveniently functionalized with the photoclick thiol-ene reaction. In addition, we show for the first time that the PAINTS bearing vinyl groups can be easily modified with disulfides under UV irradiation. The effect of superhydrophobicity of PAINTS on the formation of high-resolution surface patterns has been investigated. The developed reactive superhydrophobic coating can find applications for surface biofunctionalization using abundant thiol or disulfide bearing biomolecules, such as peptides, proteins, or antibodies.

UV-triggered dopamine polymerization: control of polymerization, surface coating, and photopatterning.

UV irradiation is demonstrated to initiate dopamine polymerization and deposition on different surfaces under both acidic and basic pH. The observed acceleration of the dopamine polymerization is explained by the UV-induced formation of reactive oxygen species that trigger dopamine polymerization. The UV-induced dopamine polymerization leads to a better control over polydopamine deposition and formation of functional polydopamine micropatterns.

Support and challenges to the melanosomal casing model based on nanoscale distribution of metals within iris melanosomes detected by X-ray fluorescence analysis.

Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X-ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery-based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis-based test of the 'casing model' predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes - including one with a Cu-rich shell surrounding a Ca/Zn-rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co-exist.

Possible mechanism of adhesion in a mica supported phospholipid bilayer.

Phospholipid bilayers supported on hydrophilic solids like silica and mica play a substantial role in fundamental studies and technological applications of phospholipid membranes. In both cases the molecular mechanism of adhesion between the bilayer and the support is of primary interest. Since the possibilities of experimental methods in this specific area are rather limited, the methods of computer simulation acquire great importance. In this paper we use the grand canonical Monte Carlo technique and an atomistic force field to simulate the behavior of a mica supported phospholipid bilayer in pure water as a function of the distance between the bilayer and the support. The simulation reveals a possible adhesion mechanism, where the adhesion is due to individual lipid molecules that protrude from the bilayer and form widely spaced links with the support. Simultaneously, the bilayer remains separated from the bilayer by a thin water interlayer which maintains the bilayer fluidity.

Direct three-dimensional imaging of polymer-water interfaces by nanoscale hard X-ray phase tomography.

We report three-dimensional (3D) direct imaging of complex surface-liquid interfaces by hard X-ray phase contrast tomography as a non-destructive approach for the morphological characterization of surfaces at the micro- and nanoscale in contact with water. Specifically, we apply this method to study the solid-air-water interface in hydrophobic macroporous polymethacrylate surfaces, and the solid-oil-water interface in slippery liquid-infused porous surfaces (SLIPS). Varying the isotropic spatial resolution allows the 3D quantitative characterization of individual polymer globules, globular clusters (porosity) as well as the infused lubricant layer on SLIPS. Surface defects were resolved at the globular level. We show the first application of X-ray nanotomography to hydrated surface characterizations and we anticipate that X-ray nanoscale imaging will open new ways for various surface/interface studies.

Direct UV-induced functionalization of surface hydroxy groups by thiol-ol chemistry.

A novel UV-initiated surface modification method for the direct functionalization of surface hydroxy groups with thiol-containing molecules (termed "thiol-ol" modification) is described. This method is based on the oxidative conjugation of thiols to hydroxy groups. We demonstrate that different thiol-containing molecules, such as fluorophores, thiol-terminated poly(ethylene glycol) (PEG-SH), and a cysteine-containing peptide, can be attached onto the surface of porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate). Direct functionalization of other hydroxy-group-bearing surfaces, fabrication of micropatterns, and double patterning have been also demonstrated using the thiol-ol method.

Nano-scale morphology of melanosomes revealed by small-angle X-ray scattering.

Melanosomes are highly specialized organelles that produce and store the pigment melanin, thereby fulfilling essential functions within their host organism. Besides having obvious cosmetic consequences--determining the color of skin, hair and the iris--they contribute to photochemical protection from ultraviolet radiation, as well as to vision (by defining how much light enters the eye). Though melanosomes can be beneficial for health, abnormalities in their structure can lead to adverse effects. Knowledge of their ultrastructure will be crucial to gaining insight into the mechanisms that ultimately lead to melanosome-related diseases. However, due to their small size and electron-dense content, physiologically intact melanosomes are recalcitrant to study by common imaging techniques such as light and transmission electron microscopy. In contrast, X-ray-based methodologies offer both high spatial resolution and powerful penetrating capabilities, and thus are well suited to study the ultrastructure of electron-dense organelles in their natural, hydrated form. Here, we report on the application of small-angle X-ray scattering--a method effective in determining the three-dimensional structures of biomolecules--to whole, hydrated murine melanosomes. The use of complementary information from the scattering signal of a large ensemble of suspended organelles and from single, vitrified specimens revealed a melanosomal sub-structure whose surface and bulk properties differ in two commonly used inbred strains of laboratory mice. Whereas melanosomes in C57BL/6J mice have a well-defined surface and are densely packed with 40-nm units, their counterparts in DBA/2J mice feature a rough surface, are more granular and consist of 60-nm building blocks. The fact that these strains have different coat colors and distinct susceptibilities to pigment-related eye disease suggest that these differences in size and packing are of biological significance.

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells.

The interaction between the stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1α axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1α levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1α significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1α interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1α gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1α axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.

Slippery liquid-infused porous surfaces showing marine antibiofouling properties.

Marine biofouling is a longstanding problem because of the constant challenges placed by various fouling species and increasingly restricted environmental regulations for antifouling coatings. Novel nonbiocidal strategies to control biofouling will necessitate a multifunctional approach to coating design. Here we show that slippery liquid-infused porous surfaces (SLIPSs) provide another possible strategy to obtaining promising antifouling coatings. Microporous butyl methacrylate-ethylene dimethacrylate (BMA-EDMA) surfaces are prepared via UV-initiated free-radical polymerization. Subsequent infusion of fluorocarbon lubricants (Krytox103, Krytox100, and Fluorinert FC-70) into the porous microtexture results in liquid-repellent slippery surfaces. To study the interaction with marine fouling organisms, settlement of zoospores of the alga Ulva linza and cypris larvae of the barnacle Balanus amphitrite is tested in laboratory assays. BMA-EDMA surfaces infused with Krytox103 and Krytox100 exhibit remarkable inhibition of settlement (attachment) of both spores and cyprids to a level comparable to that of a poly(ethylene glycol) (PEG)-terminated self-assembled monolayer. In addition, the adhesion strength of sporelings (young plants) of U. linza is reduced for BMA-EDMA surfaces infused with Krytox103 and Krytox100 compared to pristine (noninfused) BMA-EDMA and BMA-EDMA infused with Fluorinert FC-70. Immersion tests suggest a correlation between the stability of slippery coatings in artificial seawater and fouling resistance efficacy. The results indicate great potential for the application of this concept in fouling-resistant marine coatings.

Hot embossed microtopographic gradients reveal morphological cues that guide the settlement of zoospores.

Among different surface cues, the settlement of cells and larvae of marine macrofouling organisms has been found to be strongly influenced by surface microtopographies. In this article, the settlement of zoospores of the green alga Ulva linza on a surface topographic gradient has been investigated. "Honeycomb" gradient structures with feature sizes ranging from 1 to 10 µm were prepared by hot embossing, and the effect on the density of spores that attached in settlement assays was quantified. The highest density of spores was found when the size of the microstructures was similar to or larger than the size of the spores. With decreasing size of the structures, spore settlement density decreased. Interestingly, spore settlement density correlated with the Wenzel roughness of the surfaces. "Kink sites" on the surface played an important role and resembled preferred attachment positions. Furthermore, the gradients allowed the minimum pit size that the spores were able to squeeze into to be determined.

Adherent cells avoid polarization gradients on periodically poled LiTaO3 ferroelectrics.

The response of fibroblast cells to periodically poled LiTaO3 ferroelectric crystals has been studied. While fibroblast cells do not show morphological differences on the two polarization directions, they show a tendency to avoid the field gradients that occur between polarization domains of the ferroelectric. The response to the field gradients is fully established after one hour, a time at which fibroblasts form their first focal contacts. If suspension cells, with a lower tendency to establish strong surface contacts are used, no influence of the field gradients is observed.

Computer simulation of water-mediated adhesion between phospholipid bilayer and solid support functionalized with self-assembled monolayers.

An attempt is made to estimate, via computer simulation of the force-distance relation, the free energy of adhesion between a phosphatidylethanolamine bilayer and an alkanethiolate self-assembled monolayer (SAM) in aqueous medium. The simulations are performed using the grand canonical Monte Carlo technique and atomistic force fields. The bilayer adhesion free energy is predicted to be -22 ± 3 mJ/m(2) (-1.4 ± 0.2 kcal/mol) on a hydrophilic carboxyl-terminated SAM and -1 ± 1 mJ/m(2) (-0.06 ± 0.06 kcal/mol) on a hydrophobic methyl-terminated SAM.