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Polymerization driven monomer passage through monolayer chemical vapour deposition graphene
Mass transport through graphene is receiving increasing attention due to the potential for molecular sieving. Experimental studies are mostly limited to the translocation of protons, ions, and water molecules, and results for larger molecules through graphene are rare. Here, we perform controlled radical polymerization with surface-anchored self-assembled initiator monolayer in a monomer solution with single-layer graphene separating the initiator from the monomer. We demonstrate that neutral monomers are able to pass through the graphene (via native defects) and increase the graphene defects ratio (Raman ID/IG) from ca. 0.09 to 0.22. The translocations of anionic and cationic monomers through graphene are significantly slower due to chemical interactions of monomers with the graphene defects. Interestingly, if micropatterned initiator-monolayers are used, the translocations of anionic monomers apparently cut the graphene sheet into congruent microscopic structures. The varied interactions between monomers and graphene defects are further investigated by quantum molecular dynamics simulations.
Impact of the hypoxic phenotype on the uptake and efflux of nanoparticles by human breast cancer cells
Breast cancer cells adapt to the hypoxic tumoral environment by undergoing changes in metabolism, cell signalling, endo-lysosomal receptor uptake and recycling. The resulting hypoxic cell phenotype has the potential to undermine the therapeutic efficacy of nanomedicines designed for endocytic uptake and specific intracellular trafficking. The aim of this study was to examine the impact of hypoxia and simulated reperfusion on the in vitro uptake and release of nanomedicines by human breast cancer cells. Cells were exposed to a hypoxic preconditioning treatment in 1% oxygen for 6 and 24 hours to induce temporal changes in the hypoxic circuit (e.g. HIF-1α expression). The preconditioned cells were then dosed with nanoparticles for 45 or 180 minutes emulating nanomedicine access following tumor reperfusion. Hypoxic preconditioning significantly increased nanoparticle retention by up to 10% when compared to normoxic cultures, with the greatest relative difference between normoxic and hypoxic cultures occurring with a 45 minute dosing interval. Exocytosis studies indicated that the preconditioned cells had a significantly increased nanoparticle efflux (up to 9%) when compared to normoxic cells. Overall, we were able to show that hypoxic preconditioning regulates both the endocytosis and exocytosis of nanomedicines in human breast cancer cells.
In vitro studies on space-conforming self-assembling silk hydrogels as a mesenchymal stem cell-support matrix suitable for minimally invasive brain application
Advanced cell therapies require robust delivery materials and silk is a promising contender with a long clinical track record. Our aim was to optimise self-assembling silk hydrogels as a mesenchymal stem cell (MSC)-support matrix that would allow future minimally invasive brain application. We used sonication energy to programme the transition of silk (1–5% w/v) secondary structure from a random coil to a stable β-sheet configuration. This allowed fine tuning of self-assembling silk hydrogels to achieve space conformity in the absence of any silk hydrogel swelling and to support uniform cell distribution as well as cell viability. Embedded cells underwent significant proliferation over 14 days in vitro, with the best proliferation achieved with 2% w/v hydrogels. Embedded MSCs showed significantly better viability in vitro after injection through a 30G needle when the gels were in the pre-gelled versus post-gelled state. Silk hydrogels (4% w/v) with physical characteristics matching brain tissue were visualised in preliminary in vivo experiments to exhibit good space conformity in an ischemic cavity (intraluminal thread middle cerebral artery occlusion model) in adult male Sprague-Dawley rats (n = 3). This study informs on optimal MSC-hydrogel matrix conditions for minimally invasive application as a platform for future experiments targeting brain repair.
MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis
MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.
Defined Geldrop Cultures Maintain Neural Precursor Cells
Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.
Self-organized stress patterns drive state transitions in actin cortices
Biological functions rely on ordered structures and intricately controlled collective dynamics. This order in living systems is typically established and sustained by continuous dissipation of energy. The emergence of collective patterns of motion is unique to nonequilibrium systems and is a manifestation of dynamic steady states. Mechanical resilience of animal cells is largely controlled by the actomyosin cortex. The cortex provides stability but is, at the same time, highly adaptable due to rapid turnover of its components. Dynamic functions involve regulated transitions between different steady states of the cortex. We find that model actomyosin cortices, constructed to maintain turnover, self-organize into distinct nonequilibrium steady states when we vary cross-link density. The feedback between actin network structure and organization of stress-generating myosin motors defines the symmetries of the dynamic steady states. A marginally cross-linked state displays divergence-free long-range flow patterns. Higher cross-link density causes structural symmetry breaking, resulting in a stationary converging flow pattern. We track the flow patterns in the model actomyosin cortices using fluorescent single-walled carbon nanotubes as novel probes. The self-organization of stress patterns we have observed in a model system can have direct implications for biological functions.
Discovery of chitin in skeletons of non-verongiid Red Sea demosponges
Marine demosponges (Porifera: Demospongiae) are recognized as first metazoans which have developed over millions of years of evolution effective survival strategies based on unique metabolic pathways to produce both biologically active secondary metabolites and biopolymer-based stiff skeletons with 3D architecture. Up to date, among marine demosponges, only representatives of the Verongiida order have been known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within skeletons of non-verongiid demosponges Acarnus wolffgangi and Echinoclathria gibbosa collected in the Red Sea. Calcofluor white staining, FTIR and Raman analysis, ESI-MS, SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm, with strong evidence, the finding of α-chitin in the skeleton of both species. We suggest that, the finding of chitin within these representatives of Poecilosclerida order is a promising step in the evaluation of these sponges as novel renewable sources for both biologically active metabolites and chitin, which are of prospective application for pharmacology and biomedicine.
Impact of the springtail’s cuticle nanotopography on bioadhesion and biofilm formation in vitro and in the oral cavity
Springtails (Collembola) have a nanostructured cuticle. To evaluate and to understand anti-biofouling properties of springtail cuticles’ morphology under different conditions, springtails, shed cuticles and cuticle replicates were studied after incubation with protein solutions and bacterial cultures using common in vitro models. In a second step, they were exposed to human oral environment in situ in order to explore potential application in dentistry. In vitro, the cuticular structures were found to resist wetting by albumin solutions for up to 3 h and colonization by Staphylococcus epidermidis was inhibited. When exposed in the oral cavity, initial pellicle formation was of high heterogeneity: parts of the surface were coated by adsorbed proteins, others remained uncoated but exhibited locally attached, ‘bridging’, proteinaceous membranes spanning across cavities of the cuticle surface; this unique phenomenon was observed for the first time. Also the degree of bacterial colonization varied considerably. In conclusion, the springtail cuticle partially modulates bioadhesion in the oral cavity in a unique and specific manner, but it has no universal effect. Especially after longer exposure, the nanotextured surface of springtails is masked by the pellicle, resulting in subsequent bacterial colonization, and, thus, cannot effectively avoid bioadhesion in the oral cavity comprehensively. Nevertheless, the observed phenomena offer valuable information and new perspectives for the development of antifouling surfaces applicable in the oral cavity.