Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin.

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

A peptidomimetic inhibitor of matrix metalloproteinases containing a tetherable linker group.

Successful wound repair and normal turnover of the extracellular matrix relies on a balance between matrix metalloproteinases (MMPs) and their natural tissue inhibitor of metalloproteinases (TIMPs). When overexpression of MMPs and abnormally high levels of activation or low expression of TIMPs are encountered, excessive degradation of connective tissue and the formation of chronic ulcers can occur. One strategy to rebalance MMPs and TIMPs is to use inhibitors. We have designed a synthetic pseudopeptide inhibitor with an amine linker group based on a known high-affinity peptidomimetic MMP inhibitor and have demonstrated inhibition of MMP-1, -2, -3, and -9 activity in standard solutions. The inhibitor was also tethered to a polyethylene glycol hydrogel using a facile reaction between the linker unit on the inhibitor and the hydrogel precursors. After tethering, we observed inhibition of the MMPs although there was an increase in the IC50s that was attributed to poor diffusion of the MMPs into the hydrogels, reduced activity of the tethered inhibitor, or incomplete incorporation of the inhibitor into the hydrogels. When the tethered inhibitors were tested against chronic wound fluid, we observed partial inhibition in proteolytic activity suggesting this approach may prove useful in rebalancing MMPs within chronic wounds.

Modeling the adhesion of human embryonic stem cells to poly(lactic-co-glycolic acid) surfaces in a 3D environment.

Human embryonic stem cells (hESCs) have previously been cultured on three dimensional (3D) biodegradable polymer scaffolds. Although complex structures were formed from the hESCs, very little is known about the mechanism of adhesion of these cells to the surfaces of the scaffolds. In this study, we achieved the efficient adhesion of pluripotent hESCs to 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds based on our data from a novel two dimensional (2D) model that imitates the surface properties of the scaffolds. In the 2D model, single cell preparations of pluripotent hESCs adhered efficiently and predominantly to PLGA surfaces coated with laminin in comparison to collagen I, collagen IV, or fibronectin-coated surfaces. Flow cytometry analysis revealed that almost all of the pluripotent single cells expressed the integrin alpha 6, with a small percentage also expressing alpha 3ss1, which facilitates adhesion to laminin. This data was then translated into the 3D environment, with the efficient binding of single pluripotent hESCs to PLGA scaffolds coated with laminin. The utility of this system was shown by the directed differentiation of single hESCs seeded within laminin-coated scaffolds toward the endoderm lineage.

Nanoscale presentation of cell adhesive molecules via block copolymer self-assembly.

Precise control over the nanoscale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, such as differentiation and apoptosis. Here, we present the self-assembly of maleimide functionalised polystyrene-block-poly (ethylene oxide) copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By manipulating the phase separation of the functional PS-PEO block copolymer used in this study, via a simple blending technique, we alter the nanoscale (on PEO domains of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from approximately 450 to approximately 900 islands per microm2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between domains of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces have been utilised to immobilise, via a zinc chelating peptide sequence, poly-histidine tagged proteins and extracellular matrix (ECM) fragments. This method is seen as an ideal platform for investigations into the role of spatial arrangements of cell adhesion molecules and ECM molecules on cell function and, in particular, control of cell phenotype.

A blank slate? Layer-by-layer deposition of hyaluronic acid and chitosan onto various surfaces.

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called "blank slate": essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering.

In this study, we investigate the fabrication of 3D porous poly(lactic-co-glycolic acid) (PLGA) scaffolds using the thermally-induced phase separation technique. The current study focuses on the selection of alternative solvents for this process using a number of criteria, including predicted solubility, toxicity, removability and processability. Solvents were removed via either vacuum freeze-drying or leaching, depending on their physical properties. The residual solvent was tested using gas chromatography-mass spectrometry. A large range of porous, highly interconnected scaffold architectures with tunable pore size and alignment was obtained, including combined macro- and microporous structures and an entirely novel 'porous-fibre' structure. The morphological features of the most promising poly(lactic-co-glycolic acid) scaffolds were analysed via scanning electron microscopy and X-ray micro-computed tomography in both two and three dimensions. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.

Controllable surface modification of poly(lactic-co-glycolic acid) (PLGA) by hydrolysis or aminolysis I: physical, chemical, and theoretical aspects.

While biodegradable, biocompatible polyesters such as poly (lactic-co-glycolic acid) (PLGA) are popular materials for the manufacture of tissue engineering scaffolds, their surface properties are not particularly suitable for directed tissue growth. Although a number of approaches to chemically modify the PLGA surface have been reported, their applicability to soft tissue scaffolds, which combine large volumes, complex shapes, and extremely fine structures, is questionable. In this paper, we describe two wet-chemical methods, base hydrolysis and aminolysis, to introduce useful levels of carboxylic acid or primary and secondary amine groups, respectively, onto the surface of PLGA with minimal degradation. The effects of temperature, concentration, pH, and solvent type on the kinetics of these reactions are studied by following changes in the wettability of the PLGA using contact angle measurements. In addition, the treated surfaces are studied using X-ray photoelectron spectroscopy (XPS) to determine the effect on the surface chemical structure. Furthermore, we show using XPS analysis that these carboxyl and amine groups are readily activated to allow the covalent attachment of biological macromolecules.