Characterization of the Zoarces viviparus liver transcriptome using massively parallel pyrosequencing

<p>Abstract</p> <p>Background</p> <p>The teleost <it>Zoarces viviparus </it>(eelpout) lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays.</p> <p>Results</p> <p>In the present study we present the first comprehensive characterization of the <it>Zoarces viviparus </it>liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of <it>Gasterosteus aculeatus </it>(stickleback) were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis.</p> <p>Conclusion</p> <p>Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate <it>de novo </it>assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in <it>Zoarces viviparus</it>.</p

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube

<p>Abstract</p> <p>Background</p> <p>The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations.</p> <p>Methods</p> <p>A method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone.</p> <p>Results</p> <p>The baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls.</p> <p>Conclusions</p> <p>The present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.</p

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

<p>Abstract</p> <p>Background</p> <p>The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.</p> <p>Methods</p> <p>Western blot and immunohistochemistry with specific antibodies were used to characterize the expression and cellular localization of the mPRs in mouse and human tissues. Taqman (Quantitative Polymerase Chain Reaction) assays were used to quantify mRNA levels in the fallopian tubes of two different mouse models after injections with different hormones and specific antagonists.</p> <p>Results</p> <p>In the fallopian tubes of both mouse and human, the expression of mPR beta and mPR gamma proteins was exclusively found in the ciliated cells. Whereas mPR beta was found on the cilia, mPR gamma was localized at the base of the same ciliated cells, as previously reported. In gonadotropin-primed mice, both mPRs genes were down-regulated after an injection with progesterone. Treatment with estradiol rapidly down-regulated the level of mPR beta mRNA and protein in immature mice. The mPR gamma protein was down-regulated around the time of ovulation in cycling women, similar to the regulation observed in mice stimulated to ovulate via gonadotropin injections.</p> <p>Conclusion</p> <p>Our findings show the presence and hormonal regulation of two distinct mPRs associated with the cilia of the fallopian tubes in both mice and women. It is hypothesized that these receptors are involved in the control of ciliary movement and, thus, gamete transport in the fallopian tubes of mammals.</p

Approximate reasoning with fuzzy-syllogistic systems

The well known Aristotelian syllogistic system consists of 256 moods. We have found earlier that 136 moods are distinct in terms of equal truth ratios that range in τ=[0,1]. The truth ratio of a particular mood is calculated by relating the number of true and false syllogistic cases the mood matches. A mood with truth ratio is a fuzzy-syllogistic mood. The introduction of (n-1) fuzzy existential quantifiers extends the system to fuzzy-syllogistic systems nS, 1<n, of which every fuzzy-syllogistic mood can be interpreted as a vague inference with a generic truth ratio that is determined by its syllogistic structure. We experimentally introduce the logic of a fuzzy-syllogistic ontology reasoner that is based on the fuzzy-syllogistic systems nS. We further introduce a new concept, the relative truth ratio rτ=[0,1] that is calculated based on the cardinalities of the syllogistic cases

Scandinavium goeteborgense gen. nov., sp. nov., a New Member of the Family Enterobacteriaceae Isolated From a Wound Infection, Carries a Novel Quinolone Resistance Gene Variant

The family Enterobacteriaceae is a taxonomically diverse and widely distributed family containing many human commensal and pathogenic species that are known to carry transferable antibiotic resistance determinants. Characterization of novel taxa within this family is of great importance in order to understand the associated health risk and provide better treatment options. The aim of the present study was to characterize a Gram-negative bacterial strain (CCUG 66741) belonging to the family Enterobacteriaceae, isolated from a wound infection of an adult patient, in Sweden. Initial phenotypic and genotypic analyses identified the strain as a member of the family Enterobacteriaceae but could not assign it to any previously described species. The complete 16S rRNA gene sequence showed highest similarity (98.8%) to four species. Whole genome sequencing followed by in silico DNA-DNA similarity analysis and average nucleotide identity (ANI) analysis confirmed that strain CCUG 66741 represents a novel taxon. Sequence comparisons of six house-keeping genes (16S rRNA, atpD, dnaJ, gyrB, infB, rpoB) with those of the type strains of the type species of related genera within the family Enterobacteriaceae indicated that the strain embodies a novel species within the family. Phylogenomic analyses (ANI-based and core genome-based phylogeny) showed that strain CCUG 66741 forms a distinct clade, representing a novel species of a distinct, new genus within the family Enterobacteriaceae, for which the name Scandinavium goeteborgense gen. nov., sp. nov. is proposed, with CCUG 66741T as the type strain (= CECT 9823T = NCTC 14286T). S. goeteborgense CCUG 66741T carries a novel variant of a chromosomally-encoded quinolone resistance gene (proposed qnrB96). When expressed in Escherichia coli, the qnrB96 gene conferred five-fold increase in minimum inhibitory concentration against ciprofloxacin. This study highlights the importance and the utility of whole genome sequencing for pathogen identification in clinical settings.publishedVersio

A novel method to discover fluoroquinolone antibiotic resistance (qnr) genes in fragmented nucleotide sequences

<p>Abstract</p> <p>Background</p> <p>Broad-spectrum fluoroquinolone antibiotics are central in modern health care and are used to treat and prevent a wide range of bacterial infections. The recently discovered <it>qnr</it> genes provide a mechanism of resistance with the potential to rapidly spread between bacteria using horizontal gene transfer. As for many antibiotic resistance genes present in pathogens today, <it>qnr</it> genes are hypothesized to originate from environmental bacteria. The vast amount of data generated by shotgun metagenomics can therefore be used to explore the diversity of <it>qnr</it> genes in more detail.</p> <p>Results</p> <p>In this paper we describe a new method to identify <it>qnr</it> genes in nucleotide sequence data. We show, using cross-validation, that the method has a high statistical power of correctly classifying sequences from novel classes of <it>qnr</it> genes, even for fragments as short as 100 nucleotides. Based on sequences from public repositories, the method was able to identify all previously reported plasmid-mediated <it>qnr</it> genes. In addition, several fragments from novel putative <it>qnr</it> genes were identified in metagenomes. The method was also able to annotate 39 chromosomal variants of which 11 have previously not been reported in literature.</p> <p>Conclusions</p> <p>The method described in this paper significantly improves the sensitivity and specificity of identification and annotation of <it>qnr</it> genes in nucleotide sequence data. The predicted novel putative <it>qnr</it> genes in the metagenomic data support the hypothesis of a large and uncharacterized diversity within this family of resistance genes in environmental bacterial communities. An implementation of the method is freely available at <url>http://bioinformatics.math.chalmers.se/qnr/</url>.</p

Scandinavium goeteborgense gen. nov., sp. nov., a New Member of the Family Enterobacteriaceae Isolated From a Wound Infection, Carries a Novel Quinolone Resistance Gene Variant

The family Enterobacteriaceae is a taxonomically diverse and widely distributed family containing many human commensal and pathogenic species that are known to carry transferable antibiotic resistance determinants. Characterization of novel taxa within this family is of great importance in order to understand the associated health risk and provide better treatment options. The aim of the present study was to characterize a Gram-negative bacterial strain (CCUG 66741) belonging to the family Enterobacteriaceae, isolated from a wound infection of an adult patient, in Sweden. Initial phenotypic and genotypic analyses identified the strain as a member of the family Enterobacteriaceae but could not assign it to any previously described species. The complete 16S rRNA gene sequence showed highest similarity (98.8%) to four species. Whole genome sequencing followed by in silico DNA-DNA similarity analysis and average nucleotide identity (ANI) analysis confirmed that strain CCUG 66741 represents a novel taxon. Sequence comparisons of six house-keeping genes (16S rRNA, atpD, dnaJ, gyrB, infB, rpoB) with those of the type strains of the type species of related genera within the family Enterobacteriaceae indicated that the strain embodies a novel species within the family. Phylogenomic analyses (ANI-based and core genome-based phylogeny) showed that strain CCUG 66741 forms a distinct clade, representing a novel species of a distinct, new genus within the family Enterobacteriaceae, for which the name Scandinavium goeteborgense gen. nov., sp. nov. is proposed, with CCUG 66741T as the type strain (= CECT 9823T = NCTC 14286T). S. goeteborgense CCUG 66741T carries a novel variant of a chromosomally-encoded quinolone resistance gene (proposed qnrB96). When expressed in Escherichia coli, the qnrB96 gene conferred five-fold increase in minimum inhibitory concentration against ciprofloxacin. This study highlights the importance and the utility of whole genome sequencing for pathogen identification in clinical settings