In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Translation of biomechanical concepts in bone tissue engineering: from animal study to revision knee arthroplasty.

Bone defects in revision knee arthroplasty are often located in load-bearing regions. The goal of this study was to determine whether a physiologic load could be used as an in situ osteogenic signal to the scaffolds filling the bone defects. In order to answer this question, we proposed a novel translation procedure having four steps: (1) determining the mechanical stimulus using finite element method, (2) designing an animal study to measure bone formation spatially and temporally using micro-CT imaging in the scaffold subjected to the estimated mechanical stimulus, (3) identifying bone formation parameters for the loaded and non-loaded cases appearing in a recently developed mathematical model for bone formation in the scaffold and (4) estimating the stiffness and the bone formation in the bone-scaffold construct. With this procedure, we estimated that after 3 years mechanical stimulation increases the bone volume fraction and the stiffness of scaffold by 1.5- and 2.7-fold, respectively, compared to a non-loaded situation.

Motion compensation strategies in magnetic resonance imaging.

Image quality in magnetic resonance imaging (MRI) is considerably affected by motion. Therefore, motion is one of the most common sources of artifacts in contemporary cardiovascular MRI. Such artifacts in turn may easily lead to misinterpretations in the images and a subsequent loss in diagnostic quality. Hence, there is considerable research interest in strategies that help to overcome these limitations at minimal cost in time, spatial resolution, temporal resolution, and signal-to-noise ratio. This review summarizes and discusses the three principal sources of motion: the beating heart, the breathing lungs, and bulk patient movement. This is followed by a comprehensive overview of commonly used compensation strategies for these different types of motion. Finally, a summary and an outlook are provided.

SwissSidechain: a molecular and structural database of non-natural sidechains.

Amino acids form the building blocks of all proteins. Naturally occurring amino acids are restricted to a few tens of sidechains, even when considering post-translational modifications and rare amino acids such as selenocysteine and pyrrolysine. However, the potential chemical diversity of amino acid sidechains is nearly infinite. Exploiting this diversity by using non-natural sidechains to expand the building blocks of proteins and peptides has recently found widespread applications in biochemistry, protein engineering and drug design. Despite these applications, there is currently no unified online bioinformatics resource for non-natural sidechains. With the SwissSidechain database (http://www.swisssidechain.ch), we offer a central and curated platform about non-natural sidechains for researchers in biochemistry, medicinal chemistry, protein engineering and molecular modeling. SwissSidechain provides biophysical, structural and molecular data for hundreds of commercially available non-natural amino acid sidechains, both in l- and d-configurations. The database can be easily browsed by sidechain names, families or physico-chemical properties. We also provide plugins to seamlessly insert non-natural sidechains into peptides and proteins using molecular visualization software, as well as topologies and parameters compatible with molecular mechanics software.

Combining geophysical and laboratory measurements for civil Engineering.

The study of wave propagation at sonic frequency in soil leads to elasticity parameter determination. These parameters are compatible to those measured simultaneously by static loading. The acquisition of in situ elasticity parameter combined with laboratory description of the elastoplastic behaviour can lead to in situ elastoplastic curves. - L'étude de la propagation des ondes acoustiques permet la détermination des paramètres d'élasticité dans les sols. Ces paramètres sont cohérents avec des mesures statiques simultanées. L'acquisition des paramètres d'élasticité in situ associée à une description du comportement élasto-plastique mesuré en laboratoire permet d'obtenir des courbes d'élastoplasticité in situ.

SwissDock, a protein-small molecule docking web service based on EADock DSS.

Most life science processes involve, at the atomic scale, recognition between two molecules. The prediction of such interactions at the molecular level, by so-called docking software, is a non-trivial task. Docking programs have a wide range of applications ranging from protein engineering to drug design. This article presents SwissDock, a web server dedicated to the docking of small molecules on target proteins. It is based on the EADock DSS engine, combined with setup scripts for curating common problems and for preparing both the target protein and the ligand input files. An efficient Ajax/HTML interface was designed and implemented so that scientists can easily submit dockings and retrieve the predicted complexes. For automated docking tasks, a programmatic SOAP interface has been set up and template programs can be downloaded in Perl, Python and PHP. The web site also provides an access to a database of manually curated complexes, based on the Ligand Protein Database. A wiki and a forum are available to the community to promote interactions between users. The SwissDock web site is available online at http://www.swissdock.ch. We believe it constitutes a step toward generalizing the use of docking tools beyond the traditional molecular modeling community.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.