Structural basis of the inhibition of cystathionine γ-lyase from Toxoplasma gondii by propargylglycine and cysteine.

Cystathionine γ-lyase (CGL) is a PLP-dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL-catalyzed process consists of an α,γ-elimination reaction that breaks down cystathionine into cysteine, α-ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2 S). Importantly, inhibition of the enzyme and consequently of its H2 S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as Toxoplasma gondii, the causative agent of toxoplasmosis, encode a CGL enzyme (TgCGL) that almost exclusively catalyzes the canonical process, with only minor reactivity to cysteine. Interestingly, the substitution of N360 by a serine (the equivalent amino acid residue in the human enzyme) at the active site changes the specificity of TgCGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the CγS and the CßS bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme-substrate specificity, we have elucidated the crystal structures of native TgCGL and the variant TgCGL-N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor d,l-propargylglycine (PPG). Our structures reveal the binding mode of each molecule within the catalytic cavity and help explain the inhibitory behavior of cysteine and PPG. A specific inhibitory mechanism of TgCGL by PPG is proposed.

Structural insight into the unique conformation of cystathionine ß-synthase from Toxoplasma gondii.

Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.

Bioresorbable and Mechanically Optimized Nerve Guidance Conduit Based on a Naturally Derived Medium Chain Length Polyhydroxyalkanoate and Poly(ε-Caprolactone) Blend.

Severe peripheral nerve injuries represent a large clinical problem with relevant challenges such as the development of successful synthetic scaffolds as substitutes to autologous nerve grafting. Numerous studies have reported the use of polyesters and type I collagen-based nerve guidance conduits (NGCs) to promote nerve regeneration through critical nerve defects while providing protection from external factors. However, none of the commercially available hollow bioresorbable NGCs have demonstrated superior clinical outcomes to an autologous nerve graft. Hence, new materials and NGC geometries have been explored in the literature to mimic the native nerve properties and architecture. Here, we report a novel blend of a natural medium chain length polyhydroxyalkanoate (MCL-PHA) with a synthetic aliphatic polyester, poly(ε-caprolactone) (PCL), suitable for extrusion-based high-throughput manufacturing. The blend was designed to combine the excellent ability of PHAs to support the growth and proliferation of mammalian cells with the good processability of PCL. The material exhibited excellent neuroregenerative properties and a good bioresorption rate, while the extruded porous tubes exhibited similar mechanical properties to the rat sciatic nerve. The NGCs were implanted to treat a 10 mm long sciatic nerve defect in rats, where significant differences were found between thin and thick wall thickness implants, and both electrophysiological and histological data, as well as the number of recovered animals, provided superior outcomes than the well-referenced synthetic Neurolac NGC.

UV-Casting on Methacrylated PCL for the Production of a Peripheral Nerve Implant Containing an Array of Porous Aligned Microchannels.

Peripheral nerves are basic communication structures guiding motor and sensory information from the central nervous system to receptor units. Severed peripheral nerve injuries represent a large clinical problem with relevant challenges to successful synthetic nerve repair scaffolds as substitutes to autologous nerve grafting. Numerous studies reported the use of hollow tubes made of synthetic polymers sutured between severed nerve stumps to promote nerve regeneration while providing protection for external factors, such as scar tissue formation and inflammation. Few approaches have described the potential use of a lumen structure comprised of microchannels or microfibers to provide axon growth avoiding misdirection and fostering proper healing. Here, we report the use of a 3D porous microchannel-based structure made of a photocurable methacrylated polycaprolactone, whose mechanical properties are comparable to native nerves. The neuro-regenerative properties of the polymer were assessed in vitro, prior to the implantation of the 3D porous structure, in a 6-mm rat sciatic nerve gap injury. The manufactured implants were biocompatible and able to be resorbed by the host's body at a suitable rate, allowing the complete healing of the nerve. The innovative design of the highly porous structure with the axon guiding microchannels, along with the observation of myelinated axons and Schwann cells in the in vivo tests, led to a significant progress towards the standardized use of synthetic 3D multichannel-based structures in peripheral nerve surgery.

Picosecond Laser Ablation of Polyhydroxyalkanoates (PHAs): Comparative Study of Neat and Blended Material Response.

Polyhydroxyalkanoates (PHAs) have emerged as a promising biodegradable and biocompatible material for scaffold manufacturing in the tissue engineering field and food packaging. Surface modification is usually required to improve cell biocompatibility and/or reduce bacteria proliferation. Picosecond laser ablation was applied for surface micro structuring of short- and medium-chain length-PHAs and its blend. The response of each material as a function of laser energy and wavelength was analyzed. Picosecond pulsed laser modified the surface topography without affecting the material properties. UV wavelength irradiation showed halved ablation thresholds compared to VIS wavelength, revealing a greater photochemical nature of the ablation process at UV wavelength. Nevertheless, the ablation rate and, therefore, ablation efficiency did not show a clear dependence on beam wavelength. The different mechanical behavior of the considered PHAs did not lead to different ablation thresholds on each polymer at a constant wavelength, suggesting the interplay of the material mechanical parameters to equalize ablation thresholds. Blended-PHA showed a significant reduction in the ablation threshold under VIS irradiation respect to the neat PHAs. Picosecond ablation was proved to be a convenient technique for micro structuring of PHAs to generate surface microfeatures appropriate to influence cell behavior and improve the biocompatibility of scaffolds in tissue engineering.

High Throughput Manufacturing of Bio-Resorbable Micro-Porous Scaffolds Made of Poly(L-lactide-co-ε-caprolactone) by Micro-Extrusion for Soft Tissue Engineering Applications.

Porous scaffolds made of elastomeric materials are of great interest for soft tissue engineering. Poly(L-lactide-co-ε-caprolactone) (PLCL) is a bio-resorbable elastomeric copolymer with tailorable properties, which make this material an appropriate candidate to be used as scaffold for vascular, tendon, and nerve healing applications. Here, extrusion was applied to produce porous scaffolds of PLCL, using NaCl particles as a leachable agent. The effects of the particle proportion and size on leaching performance, dimensional stability, mechanical properties, and ageing of the scaffolds were analyzed. The efficiency of the particle leaching and scaffold swelling when wet were observed to be dependent on the porogenerator proportion, while the secant moduli and ultimate tensile strengths were dependent on the pore size. Porosity, swelling, and mechanical properties of the extruded scaffolds were tailorable, varying with the proportion and size of porogenerator particles and showed similar values to human soft tissues like nerves and veins (E = 7-15 MPa, σu = 7 MPa). Up to 300-mm length micro-porous PLCL tube with 400-µm thickness wall was extruded, proving extrusion as a high-throughput manufacturing process to produce tubular elastomeric bio-resorbable porous scaffolds of unrestricted length with tunable mechanical properties.

Synthetic bioresorbable poly-α-hydroxyesters as peripheral nerve guidance conduits; a review of material properties, design strategies and their efficacy to date.

Implantable tubular devices known as nerve guidance conduits (NGCs) have drawn considerable interest as an alternative to autografting in the repair of peripheral nerve injuries. At present, there exists a lack of biodegradable, biocompatible materials for the fabrication of NGCs with physical properties which suitably match the native nerve tissue. Most of the existing reports have been confined to the traditional synthetic aliphatic polyesters due to their naturally-occurring degradation by-products, suitably slow in vivo resorption timeframes and relatively diverse and tailorable range of material properties. Moreover, these thermoplastic polymers can be processed into NGCs from various methods and further tweaking of physical properties can be achieved during fabrication. Although there have been many successful reports of nerve gap repair using NGCs made from these materials, the majority have been confined to basic tubular designs across short to medium nerve gaps with at best equivalent outcomes to autografts. This article reviews the performance of poly-α-hydroxyester tubes to date (including modifications to basic hollow conduits) and is intended to aid researchers as they aim to create biomimetic NGCs capable of bridging larger nerve gaps with superior results to autografting. Based on the existing reports, a next-generation bioresorbable NGC should involve a highly flexible poly-α-hydroxyester outer tube, most suitably from a lactide-caprolactone co-polymer, with some combination of internal lumen contact guidance and bioactive neurotrophic factors. However, detailed further experimentation and an interdisciplinary approach will be required to arrive at an ideal final configuration.

Surface patterning of a novel PEG-functionalized poly-l-lactide polymer to improve its biocompatibility: Applications to bioresorbable vascular stents.

Today, research in the field of bioresorbable vascular stents (BVS) not only focusses on a new material being nontoxic but also tries to enhance its biocompatibility in terms of endothelialization potential and hemocompatibility. To this end, we used picosecond laser ablation technology as a single-step and contactless method for surface microstructuring of a bioresorbable polymer which can be utilized in stent manufacture. The method works on all materials via fast material removal, can be easily adapted for micropatterning of tubular or more complex sample shapes and scaled up by means of micropatterning of metal molds for manufacturing. Here, picosecond laser ablation was applied to a bioresorbable, biologically inactive and polyethylene glycol-modified poly-L-lactide polymer (PEGylated PLLA) to generate parallel microgrooves with varying geometries. The different patterns were thoroughly evaluated by a series of cyto- and hemocompatibility tests revealing that all surfaces were non-toxic and non-hemolytic. More importantly, patterns with 20 to 25 µm wide and 6 to 7 µm deep grooves significantly enhanced endothelial cell adhesion in comparison to samples with smaller grooves. Here, human cardiac microvascular endothelial cells were found to align along the groove direction, which is thought to encourage endothelialization of intraluminal surfaces of BVS. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 624-634, 2019.

Laser Surface Microstructuring of a Bio-Resorbable Polymer to Anchor Stem Cells, Control Adipocyte Morphology, and Promote Osteogenesis.

New strategies in regenerative medicine include the implantation of stem cells cultured in bio-resorbable polymeric scaffolds to restore the tissue function and be absorbed by the body after wound healing. This requires the development of appropriate micro-technologies for manufacturing of functional scaffolds with controlled surface properties to induce a specific cell behavior. The present report focuses on the effect of substrate topography on the behavior of human mesenchymal stem cells (MSCs) before and after co-differentiation into adipocytes and osteoblasts. Picosecond laser micromachining technology (PLM) was applied on poly (L-lactide) (PLLA), to generate different microstructures (microgrooves and microcavities) for investigating cell shape, orientation, and MSCs co-differentiation. Under certain surface topographical conditions, MSCs modify their shape to anchor at specific groove locations. Upon MSCs differentiation, adipocytes respond to changes in substrate height and depth by adapting the intracellular distribution of their lipid vacuoles to the imposed physical constraints. In addition, topography alone seems to produce a modest, but significant, increase of stem cell differentiation to osteoblasts. These findings show that PLM can be applied as a high-efficient technology to directly and precisely manufacture 3D microstructures that guide cell shape, control adipocyte morphology, and induce osteogenesis without the need of specific biochemical functionalization.