The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

Recent experimental studies have demonstrated that cellular motion can be directed by topographical gradients, such as those resulting from spatial variations in the features of a micropatterned substrate. This phenomenon, known as topotaxis, is especially prominent among cells persistently crawling within a spatially varying distribution of cell-sized obstacles. In this article we introduce a toy model of topotaxis based on active Brownian particles constrained to move in a lattice of obstacles, with space-dependent lattice spacing. Using numerical simulations and analytical arguments, we demonstrate that topographical gradients introduce a spatial modulation of the particles' persistence, leading to directed motion toward regions of higher persistence. Our results demonstrate that persistent motion alone is sufficient to drive topotaxis and could serve as a starting point for more detailed studies on self-propelled particles and cells. arXiv:1908.06078v1 [cond-mat.soft]

Due to the sequence-dependent nature of the elasticity of DNA, many protein-DNA complexes and other systems in which DNA molecules must be deformed have preferences for the type of DNA sequence they interact with. SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments and similar sequence selection experiments have been used extensively to examine the (indirect readout) sequence preferences of, e.g., nucleosomes (protein spools around which DNA is wound for compactification) and DNA rings. We show how recently developed computational and theoretical tools can be used to emulate such experiments in silico. Opening up this possibility comes with several benefits. First, it allows us a better understanding of our models and systems, specifically about the roles played by the simulation temperature and the selection pressure on the sequences. Second, it allows us to compare the predictions made by the model of choice with experimental results. We find agreement on important features between predictions of the rigid base-pair model and experimental results for DNA rings and interesting differences that point out open questions in the field. Finally, our simulations allow application of the SELEX methodology to systems that are experimentally difficult to realize because they come with high energetic costs and are therefore unlikely to form spontaneously, such as very short or overwound DNA rings.

3. Data modelling, computational biology and predictive medicine, AbstractCells encounter a wide variety of physical and chemical cues when navigating their native environments. However, their response to multiple simultaneous cues is not yet clear. In particular, the influence of topography, in the presence of a chemotactic gradient, on their migratory behavior is understudied. Here, we investigate the effects of topographical guidance on highly motile amoeboid cell migration (topotaxis) generated by asymmetrically placed micropillars. The micropillar field allows for an additional, natural chemotactic gradient in two different directions, thereby revealing the relevance of topotaxis in the presence of cell migration directed by chemical gradients (chemotaxis). Interestingly, we found that the topotactic drift generated by the pillar field is conserved during chemotaxis. We show that the drifts generated by both these cues add up linearly. A coarse-grained analysis as a function of pillar spacing subsequently revealed that the strength and direction of the topotactic drift is determined by (i) the pore size, (ii) space between pores, and (iii) the effective diffusion constant of the cells. Finally, we argue that topotaxis must be conserved during chemotaxis, as it is an emergent property of both the asymmetric properties of the pillar field and the inherent stochasticity of (biased) amoeboid migration.

The primary defence mechanism of myocytes against peroxides and peroxide-derived peroxyl and alkoxyl radicals is the glutathione redox cycle. The purpose of the present study was to increase the turnover rate of this cycle by stimulating the glutathione peroxidase catalysed reaction (2GSH-->GSSG), the glutathione reductase catalysed reaction (GSSG-->2GSH), or both. Neonatal rat heart cell cultures were subjected to a standardized protocol of oxidative stress using 80 mumol.l-1 cumene hydroperoxide (CHPO) for 0-90 min. The consequences of this protocol were described in terms of cellular concentrations of GSH, GSSG, NADPH and ATP, formation of malondialdehyde (MDA), release of GSSG and of ATP catabolites, depression of contraction frequency, cellular calcium overload, and enzyme release. Trolox-C, an analogue of vitamin E, accelerated the glutathione peroxidase reaction leading to lowering of GSH concentration and the GSH/GSSG ratio, less MDA formation, diminished negative chronotropy, delayed calcium overload, and less enzyme release. Glucose was used to accelerate the glutathione reductase reaction by supplying NADPH, leading to higher GSH concentration and a higher GSH/GSSG ratio, less MDA formation, diminished negative chronotropy, unchanged development of calcium overload, and less enzyme release. As a full turn of the glutathione redox cycle involves both the peroxidase and the reductase reactions, the combination of Trolox-C and glucose was superior to either of the two alone: 90 min following addition of CHPO together with Trolox-C and glucose, the GSH concentration and the GSH/GSSG ratio were almost normal, MDA formation was extremely low, calcium overload was markedly delayed, and enzyme release hardly occurred at all. Cells remained beating in the observation period of 30 min. We conclude that the capacity of the glutathione redox cycle to withstand oxidative stress can be increased by stimulation of either the peroxidase reaction or the reductase reaction, and that optimal redox cycling is achieved by stimulation of both reactions.

A major challenge in the use of HepG2 cell culture models for drug toxicity screening is their lack of maturity in 2D culture. 3D culture in Matrigel promotes the formation of spheroids that express liver‐relevant markers, yet they still lack various primary hepatocyte functions. Therefore, alternative matrices where chemical composition and materials properties are controlled to steer maturation of HepG2 spheroids remain desired. Herein, a modular approach is taken based on a fully synthetic and minimalistic supramolecular matrix based on squaramide synthons outfitted with a cell‐adhesive peptide, RGD for 3D HepG2 spheroid culture. Co‐assemblies of RGD‐functionalized squaramide‐based and native monomers resulted in soft and self‐recovering supramolecular hydrogels with a tunable RGD concentration. HepG2 spheroids are self‐assembled and grown (≈150 µm) within the supramolecular hydrogels with high cell viability and differentiation over 21 days of culture. Importantly, significantly higher mRNA and protein expression levels of phase I and II metabolic enzymes, drug transporters, and liver markers are found for the squaramide hydrogels in comparison to Matrigel. Overall, the fully synthetic squaramide hydrogels are proven to be synthetically accessible and effective for HepG2 differentiation showcasing the potential of this supramolecular matrix to rival and replace naturally‐derived materials classically used in high‐throughput toxicity screening.

Photochemical ligation strategies in hydrogel materials are crucial to model spatiotemporal phenomena that occur in the natural extracellular matrix. We here describe the use of cyclic 1,2-dithiolanes to cross-link with norbornene on linear poly­(ethylene glycol) polymers through UV irradiation in a rapid and byproduct-free manner, resulting in branched macromolecular architectures and hydrogel materials from low-viscosity precursor solutions. Oscillatory rheology and NMR data indicate the one-pot formation of thioether and disulfide cross-links. Spatial and temporal control of the hydrogel mechanical properties and functionality was demonstrated by oscillatory rheology and confocal microscopy. A cytocompatible response of NIH 3T3 fibroblasts was observed within these materials, providing a foothold for further exploration of this photoactive cross-linking moiety in the biomedical field.

Supramolecular materials provide unique opportunities to mimic both the structure and mechanics of the biopolymer networks that compose the extracellular matrix. However, strategies to modify their filamentous structures in space and time in 3D cell culture to study cell behavior as encountered in development and disease are lacking. We herein disclose a multicomponent squaramide-based supramolecular material whose mechanics and bioactivity can be controlled by light through co-assembly of a 1,2-dithiolane (DT) monomer that forms disulfide cross-links. Remarkably, increases in storage modulus from ∼200 Pa to >10 kPa after stepwise photo-cross-linking can be realized without an initiator while retaining colorlessness and clarity. Moreover, viscoelasticity and plasticity of the supramolecular networks decrease upon photo-irradiation, reducing cellular protrusion formation and motility when performed at the onset of cell culture. When applied during 3D cell culture, force-mediated manipulation is impeded and cells move primarily along earlier formed channels in the materials. Additionally, we show photopatterning of peptide cues in 3D using either a photomask or direct laser writing. We demonstrate that these squaramide-based filamentous materials can be applied to the development of synthetic and biomimetic 3D in vitro cell and disease models, where their secondary cross-linking enables mechanical heterogeneity and shaping at multiple length scales.