Bovine AAV transcytosis inhibition by tannic acid results in functional expression of CFTR in vitro and altered biodistribution in vivo.

Bovine adeno-associated virus (BAAV) can enter a cell either through a transcytosis or transduction pathway. We previously demonstrated that particles entering via the transcytosis pathway can be redirected to transduce the cell by blocking particle exocytosis with tannic acid (TA). To investigate whether this approach is useful in lung gene therapy applications, we tested the effect of TA on BAAV transduction in cystic fibrosis airway epithelia in vitro, and in mouse lung in vivo. Our findings suggest that BAAV transcytosis can occur in vivo and that treatment with TA reduces transcytosis and increases lung transduction. TA treatment did not impair the sorting and the activity of the BAAV expressed cystic fibrosis transmembrane regulator membrane protein.

The outcomes of pathway database computations depend on pathway ontology.

Different biological notions of pathways are used in different pathway databases. Those pathway ontologies significantly impact pathway computations. Computational users of pathway databases will obtain different results depending on the pathway ontology used by the databases they employ, and different pathway ontologies are preferable for different end uses. We explore differences in pathway ontologies by comparing the BioCyc and KEGG ontologies. The BioCyc ontology defines a pathway as a conserved, atomic module of the metabolic network of a single organism, i.e. often regulated as a unit, whose boundaries are defined at high-connectivity stable metabolites. KEGG pathways are on average 4.2 times larger than BioCyc pathways, and combine multiple biological processes from different organisms to produce a substrate-centered reaction mosaic. We compared KEGG and BioCyc pathways using genome context methods, which determine the functional relatedness of pairs of genes. For each method we employed, a pair of genes randomly selected from a BioCyc pathway is more likely to be related by that method than is a pair of genes randomly selected from a KEGG pathway, supporting the conclusion that the BioCyc pathway conceptualization is closer to a single conserved biological process than is that of KEGG.

Genome annotation errors in pathway databases due to semantic ambiguity in partial EC numbers.

We report on a new type of systematic annotation error in genome and pathway databases that results from the misinterpretation of partial Enzyme Commission (EC) numbers such as '1.1.1.-'. This error results in the assignment of genes annotated with a partial EC number to many or all biochemical reactions that are annotated with the same partial EC number. That inference is faulty because of the ambiguous nature of partial EC numbers. We have observed this type of error in multiple databases, including KEGG, VIMSS and IMG, all of which assign genes to KEGG pathways. The Escherichia coli subset of the KEGG database exhibits this error for 6.8% of its gene-reaction assignments. For example, KEGG contains 17 reactions that are annotated with EC 1.1.1.-. A group of three E.coli genes, b1580 [putative dehydrogenase, NAD(P)-binding, starvation-sensing protein], b3787 (UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase) and b0207 (2,5-diketo-D-gluconate reductase B), is assigned to 15 of those reactions, despite experimental evidence indicating different single functions for two of the three genes. Furthermore, the databases (DBs) are internally inconsistent in that the description of gene functions for genes with partial EC numbers is inconsistent with the activities implied by reactions to which the genes were assigned. We infer that these inconsistencies result from the processing used to match gene products to reactions within KEGG's metabolic pathways. These errors affect scientists who use these DBs as online encyclopedias and they affect bioinformaticists who use these DBs to train and validate newly developed algorithms.

An evidence ontology for use in pathway/genome databases.

An important emerging need in Model Organism Databases (MODs) and other bioinformatics databases (DBs) is that of capturing the scientific evidence that supports the information within a DB. This need has become particularly acute as more DB content consists of computationally predicted information, such as predicted gene functions, operons, metabolic pathways, and protein properties. This paper presents an ontology for encoding the type of support and the degree of support for DB assertions, and for encoding the literature source in which that support is reported. The ontology includes a hierarchy of 35 evidence codes for modeling different types of wet-lab and computational evidence for the existence of operons and metabolic pathways, and for gene functions. We also describe an implementation of the ontology within the Pathway Tools software environment, which is used to query and update Pathway/Genome DBs such as EcoCyc, MetaCyc, and HumanCyc.

Using functional and organizational information to improve genome-wide computational prediction of transcription units on pathway-genome databases.

MOTIVATION: The prediction of transcription units (TUs, which are similar to operons) is an important problem that has been tackled using many different approaches. The availability of complete microbial genomes has made genome-wide TU predictions possible. Pathway-genome databases (PGDBs) add metabolic and other organizational (i.e. protein complexes) information to the annotated genome, and are able to capture TU organization information. These characteristics of PGDBs make them a suitable framework for the development and implementation of TU predictors. RESULTS: We implemented a TU predictor that uses only intergenic distance and functional classification of genes to predict TU boundaries, and applied it to EcoCyc, our PGDB of Escherichia coli. To this original predictor, we added information on metabolic pathways, protein complexes and transporters, all readily available in EcoCyc, in order to generate an enhanced predictor. The enhanced predictor correctly predicted 80% of the known E.coli TUs (69% of the known operons), a moderate improvement over the original predictor's performance (75% of TUs and 65% of operons correctly predicted), demonstrating that the extra information available in the PGDB does indeed improve prediction performance. Performance of this E.coli-based predictor on a genome other than that of E.coli was tested on BsubCyc, our computationally generated PGDB for Bacillus subtilis, for which a set of 100 known operons is available. Prediction accuracy decreased substantially (46% of the known operons correctly predicted). This was due in part to missing information in BsubCyc, which prevented full use of the predictor's features. The augmented predictor has been implemented as part of our Pathway Tools software suite, and can be used to populate a PGDB with predicted TUs. AVAILABILITY: The TU predictor is included in version 7.0 of the Pathway Tools software suite. Pathway Tools 7.0 is available free of charge to academic institutions and for a fee to commercial enterprises. It runs on Sun Solaris 8, Linux and Windows. TUs predicted on the Caulobacter crescentus and Mycobacterium tuberculosis (H37Rv) genomes are available in our CauloCyc and MtbrvCyc databases, available at the BioCyc web site (http://biocyc.org). To obtain version 7.0 of Pathway Tools, follow the directions in our web site, http://biocyc.org/download.shtml.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Pathway databases: a case study in computational symbolic theories.

A pathway database (DB) is a DB that describes biochemical pathways, reactions, and enzymes. The EcoCyc pathway DB (see http://ecocyc.org) describes the metabolic, transport, and genetic-regulatory networks of Escherichia coli. EcoCyc is an example of a computational symbolic theory, which is a DB that structures a scientific theory within a formal ontology so that it is available for computational analysis. It is argued that by encoding scientific theories in symbolic form, we open new realms of analysis and understanding for theories that would otherwise be too large and complex for scientists to reason with effectively.

KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response.

Keratinocyte growth factor (KGF) regulates several functions in adult and developing lung epithelia; it causes proliferation, stimulates secretion of fluid and electrolytes, enhances repair, and may minimize injury. To gain insight into the molecular processes influenced by KGF, we applied KGF to primary cultures of well-differentiated human airway epithelia and used microarray hybridization to assess the abundance of gene transcripts. Of 7,069 genes tested, KGF changed expression levels of 910. Earlier studies showed that KGF causes epithelial proliferation, and as expected, treatment altered expression of numerous genes involved in cell proliferation. We found that KGF stimulated transepithelial Cl(-) transport, but the number of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) transcripts fell. Although transcripts for ClC-1 and ClC-7 Cl(-) channels increased, KGF failed to augment transepithelial Cl(-) transport in CF epithelia, suggesting that KGF-stimulated Cl(-) transport in differentiated airway epithelia depends on the CFTR Cl(-) channel. Interestingly, KGF decreased transcripts for many interferon (IFN)-induced genes. IFN causes trafficking of Stat dimers to the nucleus, where they activate transcription of IFN-induced genes. We found that KGF prevented the IFN-stimulated trafficking of Stat1 from the cytosol to the nucleus, suggesting a molecular mechanism for KGF-mediated suppression of the IFN-signaling pathway. These results suggest that in addition to stimulating proliferation and repair of damaged airway epithelia, KGF stimulates Cl(-) transport and may dampen the response of epithelial cells to inflammatory mediators.

Database verification studies of SWISS-PROT and GenBank.

PROBLEM STATEMENT: We have studied the relationships among SWISS-PROT, TrEMBL, and GenBank with two goals. First is to determine whether users can reliably identify those proteins in SWISS-PROT whose functions were determined experimentally, as opposed to proteins whose functions were predicted computationally. If this information was present in reasonable quantities, it would allow researchers to decrease the propagation of incorrect function predictions during sequence annotation, and to assemble training sets for developing the next generation of sequence-analysis algorithms. Second is to assess the consistency between translated GenBank sequences and sequences in SWISS-PROT and TrEMBL. RESULTS: (1) Contrary to claims by the SWISS-PROT authors, we conclude that SWISS-PROT does not identify a significant number of experimentally characterized proteins. (2) SWISS-PROT is more incomplete than we expected in that version 38.0 from July 1999 lacks many proteins from the full genomes of important organisms that were sequenced years earlier. (3) Even if we combine SWISS-PROT and TrEMBL, some sequences from the full genomes are missing from the combined dataset. (4) In many cases, translated GenBank genes do not exactly match the corresponding SWISS-PROT sequences, for reasons that include missing or removed methionines, differing translation start positions, individual amino-acid differences, and inclusion of sequence data from multiple sequencing projects. For example, results show that for Escherichia coli, 80.6% of the proteins in the GenBank entry for the complete genome have identical sequence matches with SWISS-PROT/TrEMBL sequences, 13.4% have exact substring matches, and matches for 4.1% can be found using BLAST search; the remaining 2.0% of E.coli protein sequences (most of which are ORFs) have no clear matches to SWISS-PROT/TrEMBL. Although many of these differences can be explained by the complexity of the DB, and by the curation processes used to create it, the scale of the differences is notable.

Nutrient-related analysis of pathway/genome databases.

We present an algorithm that solves two related problems in the analysis of metabolic networks stored within a pathway/genome database. (1) The Forward Propagation Problem: given a set of nutrients that are inputs to the metabolic network, what compounds will be produced by the metabolic network? (2) The Backtracking Problem: given the results of a forward propagation, and given a set of essential compounds that are not produced as a result of the forward propagation, what precursors must be supplied to produce those essential compounds? A program based on this algorithm is applied to the EcoCyc database, which is a pathway/genome database for E. coli that consists of annotated genomes and the metabolic reactions and pathways associated with the known gene products. The inputs to the program are a description of the metabolic network of an organism (EcoCyc), a set of nutrients corresponding to a known minimal growth medium, and a list of essential compounds to be produced. The program "fires" the microorganism's metabolism contained in the database and predicts all synthesized and nonsynthesized essential compounds, along with the missing precursors required to produce the latter. When applied to the EcoCyc database, the program identifies a number of missing precursors that indicate incomplete regions of the database. Thus the program results can be used to evaluate existing pathway databases like EcoCyc.

Many Genbank entries for complete microbial genomes violate the Genbank standard.

A survey of Genbank entries for complete microbial genomes reveals that the majority do not conform to the Genbank standard. Typical deviations from the Genbank standard include records with information in incorrect fields, addition of extraneous and confusing information within a field, and omission of useful fields. This situation results from two principal causes: genome centres do not submit Genbank records in the proper form and the Genbank, EMBL and DDBJ staffs do not enforce the database standards that they have defined.

The osmolyte xylitol reduces the salt concentration of airway surface liquid and may enhance bacterial killing.

The thin layer of airway surface liquid (ASL) contains antimicrobial substances that kill the small numbers of bacteria that are constantly being deposited in the lungs. An increase in ASL salt concentration inhibits the activity of airway antimicrobial factors and may partially explain the pathogenesis of cystic fibrosis (CF). We tested the hypothesis that an osmolyte with a low transepithelial permeability may lower the ASL salt concentration, thereby enhancing innate immunity. We found that the five-carbon sugar xylitol has a low transepithelial permeability, is poorly metabolized by several bacteria, and can lower the ASL salt concentration in both CF and non-CF airway epithelia in vitro. Furthermore, in a double-blind, randomized, crossover study, xylitol sprayed for 4 days into each nostril of normal volunteers significantly decreased the number of nasal coagulase-negative Staphylococcus compared with saline control. Xylitol may be of value in decreasing ASL salt concentration and enhancing the innate antimicrobial defense at the airway surface.

An evaluation of ontology exchange languages for bioinformatics.

Ontologies are specifications of the concepts in a given field, and of the relationships among those concepts. The development of ontologies for molecular-biology information and the sharing of those ontologies within the bioinformatics community are central problems in bioinformatics. If the bioinformatics community is to share ontologies effectively, ontologies must be exchanged in a form that uses standardized syntax and semantics. This paper reports on an effort among the authors to evaluate alternative ontology-exchange languages, and to recommend one or more languages for use within the larger bioinformatics community. The study selected a set of candidate languages, and defined a set of capabilities that the ideal ontology-exchange language should satisfy. The study scored the languages according to the degree to which they satisfied each capability. In addition, the authors performed several ontology-exchange experiments with the two languages that received the highest scores: OML and Ontolingua. The result of those experiments, and the main conclusion of this study, was that the frame-based semantic model of Ontolingua is preferable to the conceptual graph model of OML, but that the XML-based syntax of OML is preferable to the Lisp-based syntax of Ontolingua.

Health technology assessment in Finland.

Finland has a long tradition of supporting social programs that promote equality and the welfare state. The healthcare system is financed mainly by taxation. Everyone is insured against illness. Each of Finland's five provinces is run by a provincial government that monitors the provision of social welfare and health care. However, the municipalities actually provide the services and regulate medical equipment and regionalization of services. During the early 1990s, gross domestic product (GDP) fell dramatically, and healthcare expenditure rose to 9.4% of GDP. Due to the economy's rapid recovery, the share of healthcare expenditure has again decreased and now matches the average level of OECD countries of approximately 7.7%. The former Finnish method of central planning and norm setting has guaranteed a fairly uniform development of necessary services throughout the country and free or low-cost access. Tight central planning did not, however, create incentives to contain costs. Therefore, in the beginning of the 1990s, decision-making power was largely decentralized to the municipalities, and the principles of state subsidies were reformed. In 1995, the Finnish Office for Health Care Technology Assessment (FinOHTA) was set up as a new unit of the National Research and Development Centre for Welfare and Health (STAKES). FinOHTA is intended to function as a national central body for advancing HTA-related work in Finland, with the ultimate goal of promoting the effectiveness and efficiency of Finnish health care. At present, the importance of HTA is widely recognized in Finland, especially in the face of rising healthcare costs.

An ontology for biological function based on molecular interactions.

MOTIVATIONS: A number of important bioinformatics computations involve computing with function: executing computational operations whose inputs or outputs are descriptions of the functions of biomolecules. Examples include performing functional queries to sequence and pathway databases, and determining functional equality to evaluate algorithms that predict function from sequence. A prerequisite to computing with function is the existence of an ontology that provides a structured semantic encoding of function. Functional bioinformatics is an emerging subfield of bioinformatics that is concerned with developing ontologies and algorithms for computing with biological function. RESULTS: The article explores the notion of computing with function, and explains the importance of ontologies of function to bioinformatics. The functional ontology developed for the EcoCyc database is presented. This ontology can encode a diverse array of biochemical processes, including enzymatic reactions involving small-molecule substrates and macromolecular substrates, signal-transduction processes, transport events, and mechanisms of regulation of gene expression. The ontology is validated through its use to express complex functional queries for the EcoCyc DB. CONTACT: pkarp@ai.sri.com

Global properties of the metabolic map of Escherichia coli.

The EcoCyc database characterizes the known network of Escherichia coli small-molecule metabolism. Here we present a computational analysis of the global properties of that network, which consists of 744 reactions that are catalyzed by 607 enzymes. The reactions are organized into 131 pathways. Of the metabolic enzymes, 100 are multifunctional, and 68 of the reactions are catalyzed by >1 enzyme. The network contains 791 chemical substrates. Other properties considered by the analysis include the distribution of enzyme subunit organization, and the distribution of modulators of enzyme activity and of enzyme cofactors. The dimensions chosen for this analysis can be employed for comparative functional analysis of complete genomes.

Effect of topical nasal pharmaceuticals on sodium and chloride transport by human airway epithelia.

The human airway epithelium lines the respiratory tract from the nasal mucosa to the bronchioles. Electrolyte transport by these epithelia is crucial in maintaining the appropriate volume and salt composition of the airway surface fluid. When this epithelium becomes functionally impaired, the airways are more prone to respiratory infections. We studied the effect of six common topical agents that are commonly used to treat rhinorrhea and nasal inflammation on the transepithelial resistance, sodium, and chloride transport of primary cultures of human airway epithelia grown at the air-liquid interface. The pharmaceuticals fluticasone propionate, cromolyn sodium, ipratropium bromide, azelastine, oxymetazoline, and normal saline were used and the electrical function of the epithelia was studied in Ussing chambers. Azelastin and ipratropium bromide-treated epithelia were found to have a significant decrease in transepithelial resistance. Both normal saline and fluticasone propionate resulted in significant increases in amiloride-sensitive short circuit currents that reflect sodium transport. Finally, normal saline resulted in a significant increase in bumetanide-sensitive short circuit current that reflects chloride transport across the epithelia. The data presented may explain a mechanism by which some topical pharmaceuticals help reduce rhinorrhea, and may point to some unwanted side effects of some pharmaceuticals on the electrolyte transport of the airway epithelia. In summary, several of the common topical nasal agents alter the electrolyte transport of the nasal airway epithelia. The in vivo significance of these findings is to be determined.

The EcoCyc and MetaCyc databases.

EcoCyc is an organism-specific Pathway/Genome Database that describes the metabolic and signal-transduction pathways of Escherichia coli, its enzymes, and-a new addition-its transport proteins. MetaCyc is a new metabolic-pathway database that describes pathways and enzymes of many different organisms, with a microbial focus. Both databases are queried using the Pathway Tools graphical user interface, which provides a wide variety of query operations and visualization tools. EcoCyc and MetaCyc are available at http://ecocyc.PangeaSystems.com/ecocyc/

Integrated pathway-genome databases and their role in drug discovery.

Integrated pathway-genome databases describe the genes and genome of an organism, as well as its predicted pathways, reactions, enzymes and metabolites. In conjunction with visualization and analysis software, these databases provide a framework for improved understanding of microbial physiology and for antimicrobial drug discovery. We describe pathway-based analyses of the genomes of a number of medically relevant microorganisms and a novel software tool that visualizes gene-expression data on a diagram showing the whole metabolic network of the microorganism.

Eco Cyc: encyclopedia of Escherichia coli genes and metabolism.

The EcoCyc database describes the genome and gene products of Escherichia coli, its metabolic and signal-transduction pathways, and its tRNAs. The database describes 4391 genes of E.coli, 695 enzymes encoded by a subset of these genes, 904 metabolic reactions that occur in E.coli, and the organization of these reactions into 129 metabolic pathways. The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways. EcoCyc has many references to the primary literature, and is a (qualitative) computational model of E. coli metabolism. EcoCyc is available at URL http://ecocyc. PangeaSystems.com/ecocyc/