Tolerance of formulas containing prebiotics in healthy, term infants.

OBJECTIVE: The aim of the study was to compare infants' gastrointestinal tolerance of formulas supplemented with 2 different levels of galacto-oligosaccharides (GOS) versus a control formula (CF) or human milk. METHODS: Healthy, full-term infants (n = 180) were enrolled in this 3-group controlled, double-blind, multicenter study, and a concurrently enrolled, nonrandomized human milk-fed group (HM) by 8 days of age. Infants were randomized to be fed formula supplemented with either 4 g (EF4) or 8 g (EF8) GOS/L or a CF until day of life (DOL) 119. Infants were to be seen at DOL 14, 35, 56, 84, and 119. Parents were to record detailed 24-hour information about intake, tolerance to feedings, and stool patterns and consistency each day from enrollment to DOL 35, and for 3 days before DOL 56, 84, and 119. Stool consistency was scored on a 5-point scale as watery (1), loose/mushy, soft, formed, or hard (5). RESULTS: The mean stool consistency score was higher in the CF group throughout the study (CF >  EF8 and CF > HM for all study periods and CF > EF4 from DOL 15 to 35, P < 0.05). There was a significantly higher percentage of watery stools in the EF8 versus the CF group from study day 1 (SD 1) to DOL 14 (P < 0.05), but no differences between the groups in number of stools per day. The percentage of feedings with spit up and/or vomiting within 1 hour after feeding was significantly lower for HM versus EF8 and CF from SD 1 to DOL 14 (P < 0.05). CONCLUSIONS: In this clinical study, milk-based term infant formula (Similac Advance) with 4 g GOS/L was well-tolerated in terms of stool consistency and additional measures of gastrointestinal tolerance by newborn infants through the first 4 months of life.

Identification of conserved core xylem gene sets: conifer cDNA microarray development, transcript profiling and computational analyses.

One approach for investigating the molecular basis of wood formation is to integrate microarray profiling data sets and sequence analyses, comparing tree species with model plants such as Arabidopsis. Conifers may be included in comparative studies thanks to large-scale expressed sequence tag (EST) analyses, which enable the development of cDNA microarrays with very significant genome coverage. A microarray of 10,400 low-redundancy sequences was designed starting from white spruce (Picea glauca (Moench.) Voss) cDNAs. Computational procedures that were developed to ensure broad transcriptome coverage and efficient PCR amplification were used to select cDNA clones, which were re-sequenced in the microarray manufacture process. White spruce transcript profiling experiments that compared secondary xylem to phloem and needles identified 360 xylem-preferential gene sequences. The functional annotations of all differentially expressed sequences were highly consistent with the results of similar analyses carried out in angiosperm trees and herbaceous plants. Computational analyses comparing the spruce microarray sequences and core xylem gene sets from Arabidopsis identified 31 transcripts that were highly conserved in angiosperms and gymnosperms, in terms of both sequence and xylem expression. Several other spruce sequences have not previously been linked to xylem differentiation (including genes encoding TUBBY-like domain proteins (TLPs) and a gibberellin insensitive (gai) gene sequence) or were shown to encode proteins of unknown function encompassing diverse conserved domains of unknown function.

ForestTreeDB: a database dedicated to the mining of tree transcriptomes.

ForestTreeDB is intended as a resource that centralizes large-scale expressed sequence tag (EST) sequencing results from several tree species (http://foresttree.org/ftdb). It currently encompasses 344,878 quality sequences from 68 libraries, from diverse organs of conifer and hybrid poplar trees. It utilizes the Nimbus data model to provide a hosting system for multiple projects, and uses object-relational mapping APIs in Java and Perl for data accesses within an Oracle database designed to be scalable, maintainable and extendable. Transcriptome builds or unigene sets occupy the focal point of the system. Several of the five current species-specific unigenes were used to design microarrays and SNP resources. The ForestTreeDB web application provides the means for multiple combination database queries. It presents the user with a list of discrete queries to retrieve and download large EST datasets or sequences from precompiled unigene assemblies. Functional annotation assignment is not trivial in conifers which are distantly related to angiosperm model plants. Optimal annotations are achieved through database queries that integrate results from several procedures based open-source tools. ForestTreeDB aims to facilitate sequence mining of coherent annotations in multiple species to support comparative genomic approaches. We plan to continuously enrich ForestTreeDB with other resources through collaborations with other genomic projects.

Legume Resources: MtDB and Medicago.Org.

To identify the genes and gene functions that underlie key aspects of legume biology, researchers have selected the cool season legume Medicago truncatula as a model system for legume research. The mission of the M. truncatula Consortium is to promote unrestricted sharing of data and information that are provided by Medicago research groups worldwide. Through integration of a variety of data and tools, the medicago.org site intends to facilitate progress in the fields of structural, comparative, and functional genomics. To this goal, and as a consortium partner, the Center for Computational Genomics and Bioinformatics (CCGB) at the University of Minnesota has developed MtDB2.0, the M. truncatula database version 2.0. The MtDB2.0 database is the first step toward the global integration of M. truncatula genomic, genetic, and biological information. MtDB2.0 is a relational database that integrates M. truncatula transcriptome data and provides a wide range of user-defined data mining options. The database is interrogated through a series of interfaces, with 58 options grouped into two filters. Sequence identifiers from all public M. truncatula sites [e.g., IDs from GenBank, CCGB, The Institute for Genomic Research (TIGR), National Center for Genome Resources (NCGR), and I'Institut National de la Recherche Agronomique (INRA)] are fully cross-referenced to facilitate comparisons between different sites, and hypertext links to the appropriate database records are provided for all queries' results. MtDB's goal is to provide researchers with the means to quickly and independently identify sequences that match specific research interests based on user-defined criteria. MtDB2.0 offers unrestricted access to advanced and powerful querying tools unmatched by any other public databases. Structurad Query Language (SQL)-encoded queries with a Java-based Web user interface, incorporate different filtering that allow sophisticated data mining of the expressed sequence tag sequencing project results, including the CCGB M. truncatula Unigene set generated with the Phrap assembler. The underlying database and query software have been designed for ease of updates and portability to other model organisms. Public access to the database is at http://www.medicago.org/MtDB.

Automated SNP detection from a large collection of white spruce expressed sequences: contributing factors and approaches for the categorization of SNPs.

BACKGROUND: High-throughput genotyping technologies represent a highly efficient way to accelerate genetic mapping and enable association studies. As a first step toward this goal, we aimed to develop a resource of candidate Single Nucleotide Polymorphisms (SNP) in white spruce (Picea glauca [Moench] Voss), a softwood tree of major economic importance. RESULTS: A white spruce SNP resource encompassing 12,264 SNPs was constructed from a set of 6,459 contigs derived from Expressed Sequence Tags (EST) and by using the bayesian-based statistical software PolyBayes. Several parameters influencing the SNP prediction were analysed including the a priori expected polymorphism, the probability score (PSNP), and the contig depth and length. SNP detection in 3' and 5' reads from the same clones revealed a level of inconsistency between overlapping sequences as low as 1%. A subset of 245 predicted SNPs were verified through the independent resequencing of genomic DNA of a genotype also used to prepare cDNA libraries. The validation rate reached a maximum of 85% for SNPs predicted with either PSNP > or = 0.95 or > or = 0.99. A total of 9,310 SNPs were detected by using PSNP > or = 0.95 as a criterion. The SNPs were distributed among 3,590 contigs encompassing an array of broad functional categories, with an overall frequency of 1 SNP per 700 nucleotide sites. Experimental and statistical approaches were used to evaluate the proportion of paralogous SNPs, with estimates in the range of 8 to 12%. The 3,789 coding SNPs identified through coding region annotation and ORF prediction, were distributed into 39% nonsynonymous and 61% synonymous substitutions. Overall, there were 0.9 SNP per 1,000 nonsynonymous sites and 5.2 SNPs per 1,000 synonymous sites, for a genome-wide nonsynonymous to synonymous substitution rate ratio (Ka/Ks) of 0.17. CONCLUSION: We integrated the SNP data in the ForestTreeDB database along with functional annotations to provide a tool facilitating the choice of candidate genes for mapping purposes or association studies.

Generation, annotation, analysis and database integration of 16,500 white spruce EST clusters.

BACKGROUND: The sequencing and analysis of ESTs is for now the only practical approach for large-scale gene discovery and annotation in conifers because their very large genomes are unlikely to be sequenced in the near future. Our objective was to produce extensive collections of ESTs and cDNA clones to support manufacture of cDNA microarrays and gene discovery in white spruce (Picea glauca [Moench] Voss). RESULTS: We produced 16 cDNA libraries from different tissues and a variety of treatments, and partially sequenced 50,000 cDNA clones. High quality 3' and 5' reads were assembled into 16,578 consensus sequences, 45% of which represented full length inserts. Consensus sequences derived from 5' and 3' reads of the same cDNA clone were linked to define 14,471 transcripts. A large proportion (84%) of the spruce sequences matched a pine sequence, but only 68% of the spruce transcripts had homologs in Arabidopsis or rice. Nearly all the sequences that matched the Populus trichocarpa genome (the only sequenced tree genome) also matched rice or Arabidopsis genomes. We used several sequence similarity search approaches for assignment of putative functions, including blast searches against general and specialized databases (transcription factors, cell wall related proteins), Gene Ontology term assignation and Hidden Markov Model searches against PFAM protein families and domains. In total, 70% of the spruce transcripts displayed matches to proteins of known or unknown function in the Uniref100 database (blastx e-value < 1e-10). We identified multigenic families that appeared larger in spruce than in the Arabidopsis or rice genomes. Detailed analysis of translationally controlled tumour proteins and S-adenosylmethionine synthetase families confirmed a twofold size difference. Sequences and annotations were organized in a dedicated database, SpruceDB. Several search tools were developed to mine the data either based on their occurrence in the cDNA libraries or on functional annotations. CONCLUSION: This report illustrates specific approaches for large-scale gene discovery and annotation in an organism that is very distantly related to any of the fully sequenced genomes. The ArboreaSet sequences and cDNA clones represent a valuable resource for investigations ranging from plant comparative genomics to applied conifer genetics.

Apparent homology of expressed genes from wood-forming tissues of loblolly pine (Pinus taeda L.) with Arabidopsis thaliana.

Pinus taeda L. (loblolly pine) and Arabidopsis thaliana differ greatly in form, ecological niche, evolutionary history, and genome size. Arabidopsis is a small, herbaceous, annual dicotyledon, whereas pines are large, long-lived, coniferous forest trees. Such diverse plants might be expected to differ in a large number of functional genes. We have obtained and analyzed 59,797 expressed sequence tags (ESTs) from wood-forming tissues of loblolly pine and compared them to the gene sequences inferred from the complete sequence of the Arabidopsis genome. Approximately 50% of pine ESTs have no apparent homologs in Arabidopsis or any other angiosperm in public databases. When evaluated by using contigs containing long, high-quality sequences, we find a higher level of apparent homology between the inferred genes of these two species. For those contigs 1,100 bp or longer, approximately 90% have an apparent Arabidopsis homolog (E value < 10-10). Pines and Arabidopsis last shared a common ancestor approximately 300 million years ago. Few genes would be expected to retain high sequence similarity for this time if they did not have essential functions. These observations suggest substantial conservation of gene sequence in seed plants.