ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

Decoupled Associative and Dissociative Processes in Strong yet Highly Dynamic Host-Guest Complexes.

Kinetics and thermodynamics in supramolecular systems are intimately linked, yet both are independently important for application in sensing assays and stimuli-responsive switching/self-healing of materials. Host-guest interactions are of particular interest in many water-based materials, sensing, and drug delivery applications. Herein we investigate the binding dynamics of a variety of electron-rich aromatic moieties forming hetero-ternary complexes with the macrocycle cucurbit[8]uril (CB[8]) and an auxiliary guest, dimethyl viologen, with high selectivity and equilibrium binding constants (Keq up to 1014 M-2). Using stopped-flow spectrofluorimetry, association rate constants were observed to approach the diffusion limit and were found to be insensitive to the structure of the guest. Conversely, the dissociation rate constants of the ternary complexes varied dramatically with the guest structure and were correlated with the thermodynamic binding selectivity. Hence differing molecular features were found to contribute to the associative and dissociative processes, mimicking naturally occurring reactions and giving rise to a decoupling of these kinetic parameters. Moreover, we demonstrate the ability to exploit these phenomena and selectively perturb the associative process with external stimuli (e.g., viscosity and pressure). Significantly, these complexes exhibit increased binding equilibria with increasing pressure, with important implications for the application of the CB[8] ternary complex for the formation of hydrogels, as these gels exhibit unprecedented pressure-insensitive rheological properties. A high degree of flexibility therefore exists in the design of host-guest systems with tunable kinetic and thermodynamic parameters for tailor-made applications across a broad range of fields.

Preparation and Supramolecular Recognition of Multivalent Peptide-Polysaccharide Conjugates by Cucurbit[8]uril in Hydrogel Formation.

Supramolecular hydrogels were fabricated by physically cross-linking phenylalanine functionalized polysaccharides with cucurbit[8]uril in water. We report a facile 2-step method of functionalization of the polysaccharides hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), and guar with the dipeptide Phe-Cys. Addition of cucurbit[8]uril to the functional polysaccharides initiated physical cross-linking on account of strong 1:2 "homoternary" complexes with the pendant Phe residues. In particular, HA and CMC based soft hydrogels displayed impressive viscoelastic behavior which was characterized using rheology, demonstrating accessibility to an array of material properties which would find broad applicability in many fields.

Masking the transmembrane region of the amyloid ß precursor protein as a safe means to lower amyloid ß production.

Introduction: Reducing brain levels of both soluble and insoluble forms of amyloid beta (Aß) remains the primary goal of most therapies that target Alzheimer's disease (AD). However, no treatment has so far resulted in patient benefit, and clinical trials of the most promising drug candidates have generally failed due to significant adverse effects. This highlights the need for safer and more selective ways to target and modulate Aß biogenesis. Methods: Peptide technology has advanced to allow reliable synthesis, purification, and delivery of once-challenging hydrophobic sequences. This is opening up new routes to target membrane processes associated with disease. Here we deploy a combination of atomic detail molecular dynamics (MD) simulations, living-cell Förster resonance energy transfer (FRET), and in vitro assays to elucidate the atomic-detail dynamics, molecular mechanisms, and cellular activity and selectivity of a membrane-active peptide that targets the Aß precursor protein (APP). Results: We demonstrate that Aß biogenesis can be downregulated selectively using an APP occlusion peptide (APPOP). APPOP inhibits Aß production in a dose-dependent manner, with a mean inhibitory concentration (IC50) of 450 nM toward exogenous APP and 50 nM toward endogenous APP in primary rat cortical neuronal cultures. APPOP does not impact the γ-secretase cleavage of Notch-1, or exhibit toxicity toward cultured primary rat neurons, suggesting that it selectively shields APP from proteolysis. Discussion: Drugs targeting AD need to be given early and for very long periods to prevent the onset of clinical symptoms. This necessitates being able to target Aß production precisely and without affecting the activity of key cellular enzymes such as γ-secretase for other substrates. Peptides offer a powerful way for targeting key pathways precisely, thereby reducing the risk of adverse effects. Here we show that protecting APP from proteolytic processing offers a promising route to safely and specifically lower Aß burden. In particular, we show that the amyloid pathway can be targeted directly and specificically. This reduces the risk of off-target effects and paves the way for a safe prophylactic treatment.

Selectively Cross-Linked Tetra-PEG Hydrogels Provide Control over Mechanical Strength with Minimal Impact on Diffusivity.

Synthetic hydrogels formed from poly(ethylene glycol) (PEG) are widely used to study how cells interact with their extracellular matrix. These in vivo-like 3D environments provide a basis for tissue engineering and cell therapies but also for research into fundamental biological questions and disease modeling. The physical properties of PEG hydrogels can be modulated to provide mechanical cues to encapsulated cells; however, the impact of changing hydrogel stiffness on the diffusivity of solutes to and from encapsulated cells has received only limited attention. This is particularly true in selectively cross-linked "tetra-PEG" hydrogels, whose design limits network inhomogeneities. Here, we used a combination of theoretical calculations, predictive modeling, and experimental measurements of hydrogel swelling, rheological behavior, and diffusion kinetics to characterize tetra-PEG hydrogels' permissiveness to the diffusion of molecules of biologically relevant size as we changed polymer concentration, and thus hydrogel mechanical strength. Our models predict that hydrogel mesh size has little effect on the diffusivity of model molecules and instead predicts that diffusion rates are more highly dependent on solute size. Indeed, our model predicts that changes in hydrogel mesh size only begin to have a non-negligible impact on the concentration of a solute that diffuses out of hydrogels for the smallest mesh sizes and largest diffusing solutes. Experimental measurements characterizing the diffusion of fluorescein isothiocyanate (FITC)-labeled dextran molecules of known size aligned well with modeling predictions and suggest that doubling the polymer concentration from 2.5% (w/v) to 5% produces stiffer gels with faster gelling kinetics without affecting the diffusivity of solutes of biologically relevant size but that 10% hydrogels can slow their diffusion. Our findings provide confidence that the stiffness of tetra-PEG hydrogels can be modulated over a physiological range without significantly impacting the transport rates of solutes to and from encapsulated cells.

Protein-mediated gelation and nano-scale assembly of unfunctionalized hyaluronic acid and chondroitin sulfate.

Background: Hyaluronic acid (HA) is a major component of the extracellular matrix (ECM) in the central nervous system and the only purely supramolecular glycosaminoglycan. Much focus has been given to using this high molecular weight polysaccharide for tissue engineering applications. In most studies, the backbone of HA is functionalized with moieties that can facilitate network formation through physical self-assembly, or covalent crosslinking (e.g. photo-catalyzed) at concentrations where the polysaccharide does not gel on its own. However, these crosslinks often utilize functional groups not found in biological tissues. Methods: Oscillatory rheology, dynamic light scattering, and scanning electron microscopy were used to study albumin/HA structures. Dynamic light scattering and transmission electron microscopy were used to study albumin/chondroitin sulfate (CS) structures. UV-vis spectroscopy was used to demonstrate the potential for using protein-polymer blends as an ECM-mimetic model to study transport of small molecules. Results: We examine the intermolecular interactions of two major glycosaminoglycans found in the human brain, HA and the lower molecular weight CS, with the model protein albumin. We report the properties of the resulting micro- and nano materials. Our albumin/HA systems formed gels, and albumin/CS systems formed micro- and nanoparticles. These systems are formed from unfunctionalized polysaccharides, which is an attractive and simple method of forming HA hydrogels and CS nanoparticles. We also summarize the concentrations of HA and CS found in various mammalian brains, which could potentially be useful for biomimetic scaffold development. Conclusions: Simple preparation of commercially available charged biomacromolecules results in interesting materials with structures at the micron and nanometer length-scales. Such materials may have utility in serving as cost-effective models of nervous system electrostatic interactions and as in vitro drug release and model system for ECM transport studies.