In silico analysis of expressed sequence tags from Trichostrongylus vitrinus (Nematoda): comparison of the automated ESTExplorer workflow platform with conventional database searches

<p>Abstract</p> <p>Background</p> <p>The analysis of expressed sequence tags (EST) offers a rapid and cost effective approach to elucidate the transcriptome of an organism, but requires several computational methods for assembly and annotation. Researchers frequently analyse each step manually, which is laborious and time consuming. We have recently developed ESTExplorer, a semi-automated computational workflow system, in order to achieve the rapid analysis of EST datasets. In this study, we evaluated EST data analysis for the parasitic nematode <it>Trichostrongylus vitrinus </it>(order Strongylida) using ESTExplorer, compared with database matching alone.</p> <p>Results</p> <p>We functionally annotated 1776 ESTs obtained <it>via </it>suppressive-subtractive hybridisation from <it>T. vitrinus</it>, an important parasitic trichostrongylid of small ruminants. Cluster and comparative genomic analyses of the transcripts using ESTExplorer indicated that 290 (41%) sequences had homologues in <it>Caenorhabditis elegans</it>, 329 (42%) in parasitic nematodes, 202 (28%) in organisms other than nematodes, and 218 (31%) had no significant match to any sequence in the current databases. Of the <it>C. elegans </it>homologues, 90 were associated with 'non-wildtype' double-stranded RNA interference (RNAi) phenotypes, including embryonic lethality, maternal sterility, sterile progeny, larval arrest and slow growth. We could functionally classify 267 (38%) sequences using the Gene Ontologies (GO) and establish pathway associations for 230 (33%) sequences using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Further examination of this EST dataset revealed a number of signalling molecules, proteases, protease inhibitors, enzymes, ion channels and immune-related genes. In addition, we identified 40 putative secreted proteins that could represent potential candidates for developing novel anthelmintics or vaccines. We further compared the automated EST sequence annotations, using ESTExplorer, with database search results for individual <it>T. vitrinus </it>ESTs. ESTExplorer reliably and rapidly annotated 301 ESTs, with pathway and GO information, eliminating 60 low quality hits from database searches.</p> <p>Conclusion</p> <p>We evaluated the efficacy of ESTExplorer in analysing EST data, and demonstrate that computational tools can be used to accelerate the process of gene discovery in EST sequencing projects. The present study has elucidated sets of relatively conserved and potentially novel genes for biological investigation, and the annotated EST set provides further insight into the molecular biology of <it>T. vitrinus</it>, towards the identification of novel drug targets.</p

Amplified biochemical oscillations in cellular systems

We describe a mechanism for pronounced biochemical oscillations, relevant to microscopic systems, such as the intracellular environment. This mechanism operates for reaction schemes which, when modeled using deterministic rate equations, fail to exhibit oscillations for any values of rate constants. The mechanism relies on amplification of the underlying stochasticity of reaction kinetics within a narrow window of frequencies. This amplification allows fluctuations to beat the central limit theorem, having a dominant effect even though the number of molecules in the system is relatively large. The mechanism is quantitatively studied within simple models of self-regulatory gene expression, and glycolytic oscillations.Comment: 35 pages, 6 figure

Genotypic and phenotypic characterization of enteric bacteria in an integrated population of swine and humans

In two longitudinal studies, we examined the transmission dynamics of antimicrobial resistance (AMR) in an integrated, semi-closed population of humans and swine

Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum

Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism

Identifying risk and mitigating gambling-related harm in online poker

The present paper conducts a critical analysis of the potential for gambling-related harm in relation to online poker participation, and a theoretical evaluation of current responsible gambling strategies employed to mitigate harm in online gambling and applies the evaluation of these strategies specifically to online poker gambling. Theoretically, the primary risk for harm in online poker is the rapid and continuous nature of poker provisions online, and has been demonstrated to be associated with disordered gambling behaviour, including the chasing of monetary losses. The following responsible gambling features were deemed relevant for consideration: informed player choice, voluntary self-exclusion, employee intervention, pre-commitment, in-game feedback, behavioural tracking tools, and age restriction and verification. Although current responsible gambling features are evaluated as theoretically robust, there remains a fundamental need for experimental validation of their effectiveness. Furthermore, despite online poker gamblers perceiving the responsible gambling features as valuable tools, in reality very few players regularly use available responsible gambling features. Ultimately, for the online poker gambling industry to retain market credibility and avoid substantial top-down regulation, it is imperative to demonstrate effectiveness of responsible gambling approaches, and increase customer utilisation of available harm-mitigation features

Real-time analysis of δ13C- and δD-CH4 in ambient air with laser spectroscopy:method development and first intercomparison results

In situ and simultaneous measurement of the three most abundant isotopologues of methane using mid-infrared laser absorption spectroscopy is demonstrated. A field-deployable, autonomous platform is realized by coupling a compact quantum cascade laser absorption spectrometer (QCLAS) to a preconcentration unit, called trace gas extractor (TREX). This unit enhances CH4 mole fractions by a factor of up to 500 above ambient levels and quantitatively separates interfering trace gases such as N2O and CO2. The analytical precision of the QCLAS isotope measurement on the preconcentrated (750 ppm, parts-per-million, µmole mole−1) methane is 0.1 and 0.5 ‰ for δ13C- and δD-CH4 at 10 min averaging time. Based on repeated measurements of compressed air during a 2-week intercomparison campaign, the repeatability of the TREX–QCLAS was determined to be 0.19 and 1.9 ‰ for δ13C and δD-CH4, respectively. In this intercomparison campaign the new in situ technique is compared to isotope-ratio mass spectrometry (IRMS) based on glass flask and bag sampling and real time CH4 isotope analysis by two commercially available laser spectrometers. Both laser-based analyzers were limited to methane mole fraction and δ13C-CH4 analysis, and only one of them, a cavity ring down spectrometer, was capable to deliver meaningful data for the isotopic composition. After correcting for scale offsets, the average difference between TREX–QCLAS data and bag/flask sampling–IRMS values are within the extended WMO compatibility goals of 0.2 and 5 ‰ for δ13C- and δD-CH4, respectively. This also displays the potential to improve the interlaboratory compatibility based on the analysis of a reference air sample with accurately determined isotopic composition