Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation

Funding: We are grateful to A.N. Odyniec, M. Brigl, G.F.M. Watts, and T.Y. Cheng for suggestions and excellent technical assistance. This work was supported by National Institutes of Health (NIH) Grants AI028973 and AI063428 (to M.B.B.), DK36729 and NS36681 (to G.A.G.), and AR048632 and AI049313 (to D.B.M. and A.K.); a Howard Hughes Medical Institute Gilliam Fellowship (to L.L.); the Burroughs Wellcome Fund (D.B.M. and A.K.); a Personal Research Chair from Mr. James Bardrick (to V.B., N.V., and G.S.B.); a Royal Society Wolfson Research Merit Award (to V.B., N.V., and G.S.B.); the Medical Research Council (V.B., N.V., and G.S.B.); Wellcome Trust Grant 084923/B/08/Z (to V.B., N.V., and G.S.B.); and a Netherlands Organization for Scientific Research Grant (to A.J.M.Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.publishersversionpublishe

Natural carriers for application in tuberculosis treatment

Tuberculosis remains the leading cause of preventable deaths worldwide and unsuccessful therapy is mainly due to non-compliance with very prolonged treatments, often associated with severe side-effects. Overcoming this problem demands the introduction of drug carriers releasing the antimicrobial agents in a targeted and sustained manner, allowing reduction in frequency and dosing numbers. Nano and microparticles have taken the forefront of this approach, providing the means for the desired improvement of therapeutic schedules. Natural polymers are strong candidates as matrix forming materials, usually exhibiting biocompatibility, biodegradability, low cost and some technological advantages as compared with synthetic counterparts. In this review, natural particulate carriers developed for tuberculosis therapy are presented, mainly focusing on the use of polysaccharides and lipids. Their effectiveness is discussed taking into account their composition. Finally, considerations on the general potential of natural materials for this application, as well as key factors still to be addressed, are discussed

Fabrication of endothelial cell-laden carrageenan microfibers for microvascularized bone tissue engineering applications

ecent achievements in the area of tissue engineering (TE) have enabled the development of three-dimensional (3D) cell-laden hydrogels as in vitro platforms that closely mimic the 3D scenario found in native tissues. These platforms are extensively used to evaluate cellular behavior, cell-cell interactions, and tissue-like formation in highly defined settings. In this study, we propose a scalable and flexible 3D system based on microsized hydrogel fibers that might be used as building blocks for the establishment of 3D hydrogel constructs for vascularized bone TE applications. For this purpose, chitosan (CHT) coated κ-carrageenan (κ-CA) microfibers were developed using a two-step procedure involving ionotropic gelation (for the fiber formation) of κ-CA and its polyelectrolyte complexation with CHT (for the enhancement of fiber stability). The performance of the obtained fibers was assessed regarding their swelling and stability profiles, as well as their ability to carry and, subsequently, promote the outward release of microvascular-like endothelial cells (ECs), without compromising their viability and phenotype. Finally, the possibility of assembling and integrating these cell-laden fibers within a 3D hydrogel matrix containing osteoblast-like cells was evaluated. Overall, the obtained results demonstrate the suitability of the microsized κ-CA fibers to carry and deliver phenotypically apt microvascular-like ECs. Furthermore, it is shown that it is possible to assemble these cell-laden microsized fibers into 3D heterotypic hydrogels constructs. This in vitro 3D platform provides a versatile approach to investigate the interactions between multiple cell types in controlled settings, which may open up novel 3D in vitro culture techniques to better mimic the complexity of tissues.Authors thank the Portuguese Foundation for Science and Technology (FCT) for the personal grants SFRH/BD/42968/2008 through the MIT-Portugal Program (SMM) and SFRH/BD/64070/2009 (EGP). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS and MIT/ECE/0047/2009 project