Frederik Claeyssens

Chorismate mutase is at the centre of current controversy about fundamental features of biological catalysts. Some recent studies have proposed that catalysis in this enzyme does not involve transition state (TS) stabilization but instead is due largely to the formation of a reactive conformation of the substrate. To understand the origins of catalysis, it is necessary to compare equivalent reactions in different environments. The pericyclic conversion of chorismate to prephenate catalysed by chorismate mutase also occurs (much more slowly) in aqueous solution. In this study we analyse the origins of catalysis by comparison of multiple quantum mechanics/molecular mechanics (QM/MM) reaction pathways at a reliable, well tested level of theory (B3LYP/6-31G(d)/CHARMM27) for the reaction (i) in Bacillus subtilis chorismate mutase (BsCM) and (ii) in aqueous solvent. The average calculated reaction (potential energy) barriers are 11.3 kcal mol(-1) in the enzyme and 17.4 kcal mol(-1) in water, both of which are in good agreement with experiment. Comparison of the two sets of reaction pathways shows that the reaction follows a slightly different reaction pathway in the enzyme than in it does in solution, because of a destabilization, or strain, of the substrate in the enzyme. The substrate strain energy within the enzyme remains constant throughout the reaction. There is no unique reactive conformation of the substrate common to both environments, and the transition state structures are also different in the enzyme and in water. Analysis of the barrier heights in each environment shows a clear correlation between TS stabilization and the barrier height. The average differential TS stabilization is 7.3 kcal mol(-1) in the enzyme. This is significantly higher than the small amount of TS stabilization in water (on average only 1.0 kcal mol(-1) relative to the substrate). The TS is stabilized mainly by electrostatic interactions with active site residues in the enzyme, with Arg90, Arg7 and Glu78 generally the most important. Conformational effects (e.g. strain of the substrate in the enzyme) do not contribute significantly to the lower barrier observed in the enzyme. The results show that catalysis is mainly due to better TS stabilization by the enzyme.

Polymerised High Internal Phase Emulsions (PolyHIPEs) are manufactured via emulsion templating and exhibit a highly interconnected microporosity. These materials are commonly used as thin membranes for 3D cell culture. This study uses emulsion templating in combination with microstereolithography to fabricate PolyHIPE scaffolds with a tightly controlled and reproducible architecture. This combination of methods produces hierarchical structures, where the microstructural properties can be independently controlled from the scaffold macrostructure. PolyHIPEs were fabricated with varying ratios of two acrylate monomers (2-ethylhexyl acrylate (EHA) and isobornyl acrylate (IBOA)) and varying nominal porosity to tune mechanical properties. Young's modulus, ultimate tensile stress (UTS) and elongation at failure were determined for twenty EHA/IBOA compositions. Moduli ranged from 63.01±9.13 to 0.36±0.04MPa, UTS from 2.03±0.33 to 0.11±0.01MPa and failure strain from 21.86±2.87% to 2.60±0...

The plume of ejected material accompanying pulsed laser ablation of a ZnO target at 193 nm in vacuum has been investigated using wavelength and spatially resolved optical emission spectroscopy and Langmuir probes. All lines in the observed optical emission spectra are assignable to ...

For the first time a series of functional hydrogels based on semi-interpenetrating networks with both branched and crosslinked polymer components have been prepared and we show the successful use of these materials as substrates for cell culture. The materials consist of highly branched poly(N-isopropyl acrylamide)s with peptide functionalised end groups in a continuous phase of crosslinked poly(vinyl pyrrolidone). Functionalisation of the end groups of the branched polymer component with the GRGDS peptide produces a hydrogel that supports cell adhesion and proliferation. The materials provide a new synthetic functional biomaterial that has many of the features of extracellular matrix, and as such can be used to support tissue regeneration and cell culture. This class of high water content hydrogel material has important advantages over other functional hydrogels in its synthesis and does not require post-processing modifications nor are functional-monomers, which change the polymerisation process, required. Thus, the systems are amenable to large scale and bespoke manufacturing using conventional moulding or additive manufacturing techniques. Processing using additive manufacturing is exemplified by producing tubes using microstereolithography.

Multiple profiles for the reaction from chorismate to prephenate in the enzyme chorismate mutase calculated with hybrid density functional combined quantum mechanics/molecular mechanics methods (B3LYP/6-31G(d)-CHARMM27) agree well with experiment, and provide direct evidence of transition-state stabilization by this important enzyme, which is at the centre of current debates about the nature of enzyme catalysis.

Two‐photon polymerization has been employed to fabricate three‐dimensional structures using the biodegradable triblock copolymer poly (ε‐caprolactone‐co‐trimethylenecarbonate)‐b‐poly (ethylene glycol)‐b‐poly (ε‐caprolactone‐co‐trimethylenecarbonate) with 4, 4'‐bis (diethylamino) benzophenone as the photoinitiator. The fabricated structures were of good quality and had four micron resolution. Initial cytotoxicity tests show that the material does not affect cell proliferation. These studies demonstrate the potential of two‐photon ...

Diamond-like carbon (DLC) is an attractive biomaterial for coating human implantable devices. Our particular research interest is in developing DLC as a coating material for implants and electrical devices for the nervous system. We previously reported that DLC is not toxic to N2a neuroblastoma cells or primary cortical neurons and showed that phosphorus-doped DLC (P:DLC) could be used to produce patterned neuron networks. In the present study we complement and extend these findings by exploring patterning of dorsal root ganglion (DRG) explants, human neural progenitor cells (hNPC) and U-87 astroglioma cells on P:DLC. Further P:DLC data is provided to highlight that P:DLC can be used as an effective coating material for in vitro multi-electrode arrays (MEAs) with potential for patterning groups of neurons on selected electrodes. We also introduce ultraviolet (UV) irradiation as a simple treatment to render DLC neurocompatible. We show that UV:DLC can be used to support patterned and unpatterned cortical neuron growth. These findings strongly support the use of DLC as tailorable and tuneable substrate to study neural cell biology in vitro and in vivo. We conclude that DLC is a well-suited candidate material for coating implantable devices in the human nervous system.

Diamond-like carbon (DLC) has been explored as a biomaterial with potential use for coating implantable devices and surgical instruments. In this study the interaction of DLC with mammalian neuronal cells has been studied along with its modifications to improve its function as a biomaterial. We describe the use of DLC, oxidised DLC and phosphorus-doped DLC to support the growth and survival of primary central nervous system neurones and neuroblastoma cells. None of these substrates were cytotoxic and primary neurones adhered better to phosphorus-doped DLC than unmodified DLC. This property was used to culture cortical neurones in a predetermined micropattern. This raises the potential of DLC as a biomaterial for central nervous system (CNS) implantation. Furthermore, patterned DLC and phosphorus-doped DLC can direct neuronal growth, generating a powerful tool to study neuronal networks in a spatially distinct way. This study reports the generation of nerve cell patterns via patterned deposition of DLC.

In this study, we explore the production of well-defined macroscopic scaffolds with two-photon polymerization (2PP) and their use as neural tissue engineering scaffolds. We also demonstrate that these 3D scaffolds can be replicated via soft lithography, which increases production efficiency. Photopolymerizable polylactic acid (PLA) was used to produce scaffolds by 2PP and soft lithography. We assessed the biocompatibility of these scaffolds using an SH-SY5Y human neuronal cell line and primary cultured rat Schwann cells (of direct relevance to the repair of nerve injuries). A Comet assay with SH-SY5Y human neuronal cells revealed minimal DNA damage after washing the photocured material for 7 days in ethanol. Additionally, thin films and 3D scaffolds of the photocured PLA sustained a high degree of Schwann cell purity (99%), enabled proliferation over 7 days and provided a suitable substrate for supporting Schwann cell adhesion such that bi-polar and tri-polar morphologies were observed. Evidence of orthogonally aligned and organized actin thin filaments and the formation of focal contacts were observed for the majority of Schwann cells. In summary, this work supports the use of PLA as a suitable material for supporting Schwann cell growth and in turn use of 3D soft lithography for the synthesis of neural scaffolds in nerve repair.

In this study, we report the production of amine functionalized nanodiamond. The amine functionalized nanodiamond forms a conformal monolayer on a negatively charged surface produced via plasma polymerization of acrylic acid. Nanodiamond terminated surfaces were studied as substrates for neuronal cell culture. NG108-15 neuroblastoma-glioma hybrid cells were successfully cultured upon amine functionalized nanodiamond coated surfaces for between 1 and 7 d. Additionally, primary dorsal root ganglion (DRG) neurons and Schwann cells isolated from Wistar rats were also successfully cultured over a period of 21 d illustrating the potential of the coating for applications in the treatment of peripheral nerve injury.

The aim of this work was to develop an in vitro cornea model to study the effect of proinflammatory cytokines on wound healing. Initial studies investigated how to maintain the ex vivo models for up to 4 weeks without loss of epithelium. To study the effect of cytokines, corneas were cultured with the interleukins IL-17A, IL-22, or a combination of IL-17A and IL-22, or lipopolysaccharide (LPS). The effect of IL-17A on wound healing was then examined. With static culture conditions, organ cultures deteriorated within 2 weeks. With gentle rocking of media over the corneas and carbon dioxide perfusion, the ex vivo models survived for up to 4 weeks without loss of epithelium. The cytokine that caused the most damage to the cornea was IL-17A. Under static conditions, wound healing of the central corneal epithelium occurred within 9 days, but only a single-layered epithelium formed whether the cornea was exposed to IL-17A or not. With rocking of media gently over the corneas, a multilayer...

This work reports first steps towards the development of artificial neural stem cell microenvironments for the control and assessment of neural stem cell behaviour. Stem cells have been shown to be found in specific, supportive microenvironments (niches) and are believed to play an important role in tissue regeneration mechanisms. These environments are intricate spaces with chemical and biological features. Here we present work towards the development of physically defined microdevices in which neural and neural stem cells can be studied in 3-dimensions. We have approached this challenge by creating bespoke, microstructured polymer environments using both 2-photon polymerisation and soft lithography techniques. Specifically, we have designed and fabricated biodegradable microwell-shaped devices using an in house synthetized polymer (4-arm photocurable poly-lactid acid) on a bespoke 2-photon polymerisation (2PP) set-up. We have studied swelling and degradation of the constructs as well as biocompatibility. Moreover, we have explored the potential of these constructs as artificial neural cell substrates by culturing NG108-15 cells (mouse neuroblastoma; rat glioma hybrid) and human neural progenitor cells on the microstructures. Finally, we have studied the effects of our artificial microenvironments upon neurite length and cell density.