A Review of New Concepts in Renal Stone Research

Clinical and basic research in the field of urolithiasis has developed rapidly in recent years. Progress in extracorporeal shock wave lithotripsy (ESWL) and percutaneous nephrolithotomy (PNL) has brought about a revolution in the surgical treatment of urolithiasis and research at the cellular and molecular level is now expanding. In spite of these advances, however, clinical treatment of urolithiasis remains far from satisfactory. Stone recurrence in many patients cannot be predicted and is beyond control of urologists mainly because the mechanisms of stone formation are still not fully understood. It is necessary to study the process of stone formation more intensely at the cellular and molecular level, and to strengthen the links between basic and clinical research in the field. In this review, the processes involved in the formation of stones are compared with those involved in normal bio-mineralization and a model of urolithiasis is put forward based on modern systems science. Attention is concentrated on: (a) Directions of research based on physico-chemical theories of stone formation; (b) The role of renal tubular defects in urolithiasis; (c) The role of free radical reactions in stone formation; and (d) Macromolecular abnormalities and their correction

Trends and Challenges in Experimental Macromolecular Crystallography

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural database

OWL-Miner: Concept Induction in OWL Knowledge Bases

The Resource Description Framework (RDF) and Web Ontology Language (OWL) have been widely used in recent years, and automated methods for the analysis of data and knowledge directly within these formalisms are of current interest. Concept induction is a technique for discovering descriptions of data, such as inducing OWL class expressions to describe RDF data. These class expressions capture patterns in the data which can be used to characterise interesting clusters or to act as classifica- tion rules over unseen data. The semantics of OWL is underpinned by Description Logics (DLs), a family of expressive and decidable fragments of first-order logic. Recently, methods of concept induction which are well studied in the field of Inductive Logic Programming have been applied to the related formalism of DLs. These methods have been developed for a number of purposes including unsuper- vised clustering and supervised classification. Refinement-based search is a concept induction technique which structures the search space of DL concept/OWL class expressions and progressively generalises or specialises candidate concepts to cover example data as guided by quality criteria such as accuracy. However, the current state-of-the-art in this area is limited in that such methods: were not primarily de- signed to scale over large RDF/OWL knowledge bases; do not support class lan- guages as expressive as OWL2-DL; or, are limited to one purpose, such as learning OWL classes for integration into ontologies. Our work addresses these limitations by increasing the efficiency of these learning methods whilst permitting a concept language up to the expressivity of OWL2-DL classes. We describe methods which support both classification (predictive induction) and subgroup discovery (descrip- tive induction), which, in this context, are fundamentally related. We have implemented our methods as the system called OWL-Miner and show by evaluation that our methods outperform state-of-the-art systems for DL learning in both the quality of solutions found and the speed in which they are computed. Furthermore, we achieve the best ever ten-fold cross validation accuracy results on the long-standing benchmark problem of carcinogenesis. Finally, we present a case study on ongoing work in the application of OWL-Miner to a real-world problem directed at improving the efficiency of biological macromolecular crystallisation

Cyclophilin D as a multiple sclerosis and cardiovascular disease drug target

"Cyclophilins belong to the immunophilin family and are peptidyl-prolyl isomerases (PPIases) which catalyze the cis-trans interconversion of proline isomers in other proteins, an important step of protein folding. They are widely distributed in all organisms highly conserved throughout evolution. They were originally identified as the biological receptors for immunosuppressants such as Cyclosporine A, highly used to prevent immune response and organ rejection after a transplant. This immunosuppressant effect was determined to be unrelated to the intrinsic isomerase activity. They are known to be involved in pathophysiological process such as inflammation and vascular dysfunction, innate immunity to HIV, hepatitis C infection, neurodegenerative and pathological conditions and tumor biology. In particular, cyclophilin D (CypD), the mitochondrial isoform of the enzyme, is a key regulator of the mitochondrial permeability transition pore. Mitochondrial disfunction has been implicated in multiple sclerosis as well as in cardiovascular disease, suggesting CypD as a potential drug target. Following up on the increased awareness of CypD impact in human health, both pockets (proline and aniline) were screened following a Fragment-Based Drug Discovery approach at Merck KGaA (Darmstadt, Germany) where 58 confirmed fragments hits were obtained, from which only 6 CypD-fragment 3D crystal structures could be obtained.(...)

Protein regulation, protein-protein interactions and structural genomics

The new technical developments and the success of genome sequencing projects have prompted a new approach to scientific investigation and discovery in every field of biochemistry and molecular biology, including structural biology. One of the most prominent recent developments is the birth of structural genomics, a world-wide initiative that aims to provide the three-dimensional structures of all representative proteins. However, structural biology faces an exciting future beyond structural genomics; if we are to understand how the proteome works and use the genomic information for therapeutic purposes, studies of protein-protein interactions and macromolecular complexes, mechanism and regulation of macromolecular function, membrane protein structure, and structure-based therapeutic design must be pursued in parallel. Successful approaches will combine large-scale, high-throughput approaches developed through structural genomics with more traditional hypothesis-driven approaches, supported by integrative bioinformatics tools

Optimization of over-expression and purification of human leukotriene C4 synthase mutant R104A for structure-function studies by two-dimensional crystallization and electron crystallography

Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.MSCommittee Chair: Schmidt-Krey, Ingeborg; Committee Member: DiChristina, Thomas; Committee Member: Lieberman, Raque