A semantically aware platform for the authoring and secure enactment of bioinformatics workflows.

Recent advances in the field of bioinformatics present a number of challenges in the secure and efficient management and analysis of biological data resources. Workflow technologies aim to assist scientists and domain experts in the design of complex, long running, data and computing intensive experiments that involve many data processing and analysis tasks with the objective of generating new knowledge or formulate new hypothesis. In this paper we present a bioinformatics workflow authoring and execution environment that intends to greatly facilitate the whole lifecycle of such experiments. Emphasis is given on the security and ethical requirements of these scenarios and the corresponding technological response. In addition we present our semantic framework used for supporting specific user-requirements related to the reasoning and inference capabilities of the environment.

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Functional specifications of an integrated proteomics information management and analysis platform.

Detecting proteins in human blood holds the promise of a revolution in cancer diagnosis. Also, the ability to perform laboratory operations on small scales using miniaturized (lab-on-a-chip) devices has many benefits. Designing and fabricating such systems is extremely challenging, but physicists and engineers are beginning to construct such highly integrated and compact labs on chips with exciting functionality. This paper focuses on the presentation of the requirements of the information technology layer in such an integrated platform been developed in the LOCCANDIA project. LOCCANDIA is a Specific Targeted Research project (STREP) funded under the 6th Framework program of the EC. Its ultimate objective is to develop an innovative nano-technology based (lab-on-a-chip) platform for the medical-proeomics field. The paper presents the main engineering aspects, challenges and architecture for creating an Integrated Clinico-Proteomic Environment. The environment will be used to monitor and document the analysis and discovery chain and to allow the physician to interpret the digital spectrogram data delivered by the mass spectrometer, for diagnostic purposes.

Building a European biomedical grid on cancer: the ACGT Integrated Project.

This paper presents the needs and requirements that led to the formation of the ACGT (Advancing Clinico Genomic Trials) integrated project, its vision and methodological approaches of the project. The ultimate objective of the ACGT project is the development of a European biomedical grid for cancer research, based on the principles of open access and open source, enhanced by a set of interoperable tools and services which will facilitate the seamless and secure access to and analysis of multi-level clinico-genomic data, enriched with high-performing knowledge discovery operations and services. By doing so, it is expected that the influence of genetic variation in oncogenesis will be revealed, the molecular classification of cancer and the development of individualised therapies will be promoted, and finally the in-silico tumour growth and therapy response will be realistically and reliably modelled. Its main design decisions and results at its current stage of development are presented.

Improved microarray spot segmentation by combining two information channels.

High-throughput gene expression is an important aspect of modern post-genomic research. Microarray technology is the driving force of this revolution, a technology that allows the simultaneous monitoring of expression for thousands of genes. The need for accurate and reproducible research has driven the development of robust analysis frameworks for maximizing the information content of biological data. In microarray imaging technologies, several non-linearities in the experimental process render the measured expression values prone to variability and often, to poor reproducibility. Accurate segmentation of the true signal is a very important task, not least because a single value per spot needs to be derived for further knowledge discovery analysis. In this paper, we present a fully automatic segmentation method for improving the spot segmentation result. The method doesn't make any assumptions concerning the number of classes present in each image spot, and it isn't driven only by the most intense features, since it takes into account the underlying "hybridization ground truth" derived from both information channels of the spotted arrays. Our method is compared to widely used, state-of-the-art segmentation methods in microarray image analysis in a study of a metabolic disorder in yeast, where replicates of reporters are present. Initial results indicate that our method yields more reproducible log ratio measurements across replicates.

Rhizobial NodL O-acetyl transferase and NodS N-methyl transferase functionally interfere in production of modified Nod factors.

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between nodL and nodS, we have cloned the nodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.

Chitin deacetylases: new, versatile tools in biotechnology.

Chitin deacetylases have been identified in several fungi and insects. They catalyse the hydrolysis of N-acetamido bonds of chitin, converting it to chitosan. Chitosans, which are produced by a harsh thermochemical procedure, have several applications in areas such as biomedicine, food ingredients, cosmetics and pharmaceuticals. The use of chitin deacetylases for the conversion of chitin to chitosan, in contrast to the presently used chemical procedure, offers the possibility of a controlled, non-degradable process, resulting in the production of novel, well-defined chitosan oligomers and polymers.

Isolation and partial characterization of two alcohol dehydrogenase isozymes from the medfly Ceratitis capitata.

Two alcohol dehydrogenase isozymes, namely ADH-1 and ADH-2 from Ceratitis capitata were purified to homogeneity and further characterized. After ammonium sulphate precipitation from an extract of whole third instar larvae, the two isozymes were separated by ion exchange chromatography on Q-Sepharose. A combination of affinity chromatography, gel filtration and ion exchange chromatography was then used to purify each isozyme (50 and 57 times with 53 and 58% yields, for ADH-1 and -2 respectively). A crucial step for obtaining homogeneous enzyme preparations was affinity chromatography on Cibacron Blue Sepharose coupled with specific elution with NAD. Each of the isozymes is a dimer with subunit molecular weight of approximately 27 kDa. Both isozymes show a pH optimum of 9.6. ADH-1 proved to be immunochemically similar to ADH-2 when tested by Western blot analysis using polyclonal antibodies raised against ADH-1. While crude extracts of Dacus oleae ADH cross-react with these antibodies, no cross reactivity was observed with Drosophila melanogaster extracts. The sequence of a 22-residue peptide from ADH-1 was determined and showed 36% identity with residues 26-47 of the Drosophila melanogaster ADH sequence. Both the sizes of the purified proteins and the observed sequence similarity between ADH-1 and Drosophila ADH strongly suggest that the medfly ADH isozymes belong to the family of short chain dehydrogenases.

The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products.

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxii mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant similarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophilus gene product suggests that this gene encodes a polysaccharide deacetylase.

Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii.

Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine in chitin, has been purified to homogeneity from mycelial extracts of the fungus Mucor rouxii and further characterized. The enzyme exhibits a low pI (approximately 3). Its apparent molecular mass was determined to be approximately 75 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and approximately 80 kDa by size-exclusion chromatography, suggesting that the enzyme exists as a monomer. Carbohydrate analysis of purified chitin deacetylase revealed that the enzyme is a high-mannose glycoprotein and that its carbohydrate content is approximately 30% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives. The enzyme requires at least four N-acetylglucosamine residues (chitotetraose) for catalysis, and it is inhibited by carboxylic acids, particularly acetic acid. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be approximately 50 degrees C and the optimum pH was approximately 4.5.