Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro

Patterns of Genome Evolution among the Microsporidian Parasites Encephalitozoon cuniculi, Antonospora locustae and Enterocytozoon bieneusi

Microsporidia are intracellular parasites that are highly-derived relatives of fungi. They have compacted genomes and, despite a high rate of sequence evolution, distantly related species can share high levels of gene order conservation. To date, only two species have been analysed in detail, and data from one of these largely consists of short genomic fragments. It is therefore difficult to determine how conservation has been maintained through microsporidian evolution, and impossible to identify whether certain regions are more prone to genomic stasis.Here, we analyse three large fragments of the Enterocytozoon bieneusi genome (in total 429 kbp), a species of medical significance. A total of 296 ORFs were identified, annotated and their context compared with Encephalitozoon cuniculi and Antonospora locustae. Overall, a high degree of conservation was found between all three species, and interestingly the level of conservation was similar in all three pairwise comparisons, despite the fact that A. locustae is more distantly related to E. cuniculi and E. bieneusi than either are to each other.Any two genes that are found together in any pair of genomes are more likely to be conserved in the third genome as well, suggesting that a core of genes tends to be conserved across the entire group. The mechanisms of rearrangments identified among microsporidian genomes were consistent with a very slow evolution of their architecture, as opposed to the very rapid sequence evolution reported for these parasites

Monitoring and Scoring Counter-Diffusion Protein Crystallization Experiments in Capillaries by in situ Dynamic Light Scattering

In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D

Characterization of the Phytochelatin Synthase of Schistosoma mansoni

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends exclusively on the use of praziquantel. Millions of people are treated annually with praziquantel and drug resistant parasites are likely to evolve. In order to identify novel drug targets the Schistosoma mansoni sequence databases were queried for proteins involved in glutathione metabolism. One potential target identified was phytochelatin synthase (PCS). Phytochelatins are oligopeptides synthesized enzymatically from glutathione by PCS that sequester toxic heavy metals in many organisms. However, humans do not have a PCS gene and do not synthesize phytochelatins. In this study we have characterized the PCS of S. mansoni (SmPCS). The conserved catalytic triad of cysteine-histidine-aspartate found in PCS proteins and cysteine proteases is also found in SmPCS, as are several cysteine residues thought to be involved in heavy metal binding and enzyme activation. The SmPCS open reading frame is considerably extended at both the N- and C-termini compared to PCS from other organisms. Multiple PCS transcripts are produced from the single encoded gene by alternative splicing, resulting in both mitochondrial and cytoplasmic protein variants. Expression of SmPCS in yeast increased cadmium tolerance from less than 50 µM to more than 1,000 µM. We confirmed the function of SmPCS by identifying PCs in yeast cell extracts using HPLC-mass spectrometry. SmPCS was found to be expressed in all mammalian stages of worm development investigated. Increases in SmPCS expression were seen in ex vivo worms cultured in the presence of iron, copper, cadmium, or zinc. Collectively, these results indicate that SmPCS plays an important role in schistosome response to heavy metals and that PCS is a potential drug target for schistosomiasis treatment. This is the first characterization of a PCS from a parasitic organism

Notes for genera: basal clades of Fungi (including Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota)

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1--20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced

Liquid−Liquid Phase Separations in Urate Oxidase/PEG Mixtures: Characterization and Implications for Protein Crystallization

International audienceIn a previous paper (Vivares, D.; Bonnete, F. Acta Crystallogr., Sect. D 2002, 58, 472), protein-protein interactions of Aspergillus flavus urate oxidase (Uox) in solution were determined by small-angle X-ray scattering in the presence of different poly(ethylene glycol)s (PEG) in order to correlate second virial coefficient measurements with crystallization conditions. In this paper, we have characterized the experimental phase diagram of urate oxidase in the case of PEG 8000 by determining the solubility curve and the dilute part of the liquid-liquid phase separation (LLPS). Within this phase diagram, different mechanisms of urate oxidase crystal growth and LLPS can be observed by optical video microscopy. The influence of the LLPS on both the mechanisms and kinetics of urate oxidase crystal growth was observed by optical microscopy and small-angle X-ray scattering (SAXS). Interactions between the macromolecules were studied by SAXS in the dilute and dense phases of the demixed solution. It was observed that the LLPS precedes and slows down the crystallization. This study shows that urate oxidase is a good model to study protein/PEG mixtures in the general context of protein crystallization

X-ray scattering studies of Aspergillus flavus urate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins

International audienceThe determination of the three-dimensional structures of biological macromolecules by X-ray diffraction generally requires large good-quality crystals, which are often difficult to obtain as crystal nucleation and growth depend upon a great number of physicochemical parameters. In the future, the emergence of structural genomic projects will require new and rapid methods to determine crystallization conditions. Until now, the prediction of crystallization conditions has essentially been based on the knowledge of interparticular interactions in solutions inferred from studies on small soluble proteins in the presence of salts. The present study, by small-angle X-ray scattering, of urate oxidase from Aspergillus flavus, a homotetrameric enzyme of 128 kDa, allowed the extension of the results to the crystallization of large proteins in the presence of polyethylene glycol (PEG). The protein crystallization, the nucleation rate and the different morphological crystal shapes obtained were correlated with the second virial coefficient (A(2)), which was found to be in a restricted range at the low end of the 'crystallization slot' proposed by George & Wilson [(1994). Acta Cryst. D50, 361-365]

Interest of the normalized second virial coefficient and interaction potentials for crystallizing large macromolecules

International audienceIt has been shown for several years that the second virial coefficient, A(2), can be helpfully used to describe the thermodynamic behavior of biological macromolecules in solution prior to crystallization. The coefficient, which reflects either repulsive or attractive interactions between particles, can allow a rapid determination of crystallization conditions. Different biological systems, from 14 kDa to 4600 kDa, were studied by small angle Xray scattering. With large macromolecules, the A(2) values were found at the low end of the crystallization slot described by George & Wilson [(1994) Acta Cryst. D50, 361-365]. This led us to investigate the physical meaning of the second virial coefficient and to propose the use of the dimensionless second virial coefficient independent of the molecular weight and the size of the particle, which only takes into account the interaction potential between macromolecules, to predict successful crystallization conditions for large macromolecules. With this normalized coefficient (a(2)), the effect of salt on small proteins becomes equivalent to the effect of PEG on large macromolecules in terms of interaction potentials

Interactions in solution and crystallization of Aspergillus flavus urate oxidase

International audienceInterparticle interactions of urate oxidase from Aspergillus flavus have been studied by small-angle X-ray scattering to determine crystallization conditions. This enzyme is a homotetramer with a total molecular weight of 128 kDa. It is a slightly basic protein (pI between 7.5 and 8). The interaction potentials have been studied as a function of the main thermodynamic and chemical parameters: temperature, protein concentration, pH, salt nature and concentration, addition of polyols. In 10 mM sodium carbonate at pH 10.5, the interactions are slightly repulsive and become less repulsive with a pH closer to pI. With the addition of carbonate, the protein loses its tetrameric structure for a dimeric one; with formate, the tetrameric structure remains stable. We also studied the effect of polyethylene glycols as it had been done with high molecular weight proteins. With the addition of PEG 8 K, the interactions became less repulsive and even turned attractive with the addition of both PEG 8 K and salt. Protein crystals of urate oxidase were observed in slightly repulsive conditions (second virial coefficient A(2), about +10(-5) mol ml g(-2) instead of -2 to -8 x 10(-4) mol ml g(-2) for low molecular weight proteins). (C) 2001 Elsevier Science B.V. All rights reserved

Catching the PEG-induced attractive interaction between proteins

International audienceWe present the experimental and theoretical background of a method to characterize the protein-protein attractive potential induced by one of the mostly used crystallizing agents in the protein-field, the poly(ethylene glycol) (PEG). This attractive interaction is commonly called, in colloid physics, the depletion interaction. Small-Angle X-ray Scattering experiments and numerical treatments based on liquid-state theories were performed on urate oxidase-PEG mixtures with two different PEGs (3350 Da and 8000 Da). A "two-component" approach was used in which the polymer-polymer, the protein-polymer and the protein-protein pair potentials were determined. The resulting effective protein-protein potential was characterized. This potential is the sum of the free-polymer protein-protein potential and of the PEG-induced depletion potential. The depletion potential was found to be hardly dependent upon the protein concentration but strongly function of the polymer size and concentration. Our results were also compared with two models, which give an analytic expression for the depletion potential