A turn in species conservation for hairpin banksias: demonstration of oversplitting leads to better management of diversity.

PREMISE: Understanding evolutionary history and classifying discrete units of organisms remain overwhelming tasks, and lags in this workload concomitantly impede an accurate documentation of biodiversity and conservation management. Rapid advances and improved accessibility of sensitive high-throughput sequencing tools are fortunately quickening the resolution of morphological complexes and   thereby improving the estimation of species diversity. The recently described and critically endangered Banksia vincentia is morphologically similar to the hairpin banksia complex (B. spinulosa s.l.), a group of eastern Australian flowering shrubs whose continuum of morphological diversity has been responsible for taxonomic controversy and possibly questionable conservation initiatives. METHODS: To assist conservation while testing the current taxonomy of this group, we used high-throughput sequencing to infer a population-scale evolutionary scenario for a sample set that is comprehensive in its representation of morphological diversity and a 2500-km distribution. RESULTS: Banksia spinulosa s.l. represents two clades, each with an internal genetic structure shaped through historical separation by biogeographic barriers. This structure conflicts with the existing taxonomy for the group. Corroboration between phylogeny and population statistics aligns with the hypothesis that B. collina, B. neoanglica, and B. vincentia should not be classified as species. CONCLUSIONS: The pattern here supports how morphological diversity can be indicative of a locally expressed suite of traits rather than relationship. Oversplitting in the hairpin banksias is atypical since genomic analyses often reveal that species diversity is underestimated. However, we show that erring on overestimation can yield negative consequences, such as the disproportionate prioritization of a geographically anomalous population.

The impact of infectious diseases consultation on the management and outcomes of Pseudomonas aeruginosa bacteraemia in adults: a retrospective cohort study.

BACKGROUND: Pseudomonas aeruginosa bacteraemia (PAB) is associated with high mortality. The benefits of infectious diseases consultation (IDC) has been demonstrated in Staphylococcal aureus bacteraemia and other complex infections. Impact of IDC in PAB is unclear. This study aimed to evaluate the impact of IDC on the management and outcomes in patients with PAB. METHODS: This is a retrospective cohort single-centre study from 1 November 2006 to 29 May 2019, in all adult patients admitted with first episode of PAB. Data collected included demographics, clinical management and outcomes for PAB and whether IDC occurred. In addition, 29 Pseudomonas aeruginosa (PA) stored isolates were available for Illumina whole genome sequencing to investigate if pathogen factors contributed to the mortality. RESULTS: A total of 128 cases of PAB were identified, 71% received IDC. Patients who received IDC were less likely to receive inappropriate duration of antibiotic therapy (4.4%; vs 67.6%; p < 0.01), more likely to be de-escalated to oral antibiotic in a timely manner (87.9% vs 40.5%; p < 0.01), undergo removal of infected catheter (27.5% vs 13.5%; p = 0.049) and undergo surgical intervention (20.9% vs 5.4%, p = 0.023) for source control. The overall 30-day all-cause mortality rate was 24.2% and was significantly higher in the no IDC group in both unadjusted (56.8% vs 11.0%, odds ratio [OR] = 10.63, p < 0.001) and adjusted analysis (adjusted OR = 7.84; 95% confidence interval, 2.95-20.86). The genotypic analysis did not reveal any PA genetic features associated with increased mortality between IDC versus no IDC groups. CONCLUSION: Patients who received IDC for PAB had lower 30-day mortality, better source control and management was more compliant with guidelines. Further prospective studies are necessary to determine if these results can be validated in other settings.

Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs.

In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O2 or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/microRNA* species with a broad abundance range: miR-21 and miR-126 totaled almost 40% of all miRNAs. A complex repertoire of isomiRs was found, displaying also 5' variations, potentially affecting target recognition. High-stringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex. The expression of two novel miRNAs was significantly down-modulated by hypoxia, while miR-210 was significantly induced. Gene ontology analysis of their predicted targets revealed a significant association to hypoxia-inducible factor signaling, cardiovascular diseases, and cancer. Overexpression of the novel miRNAs in hypoxic endothelial cells affected cell growth and confirmed the biological relevance of their down-modulation. In conclusion, deep-sequencing accurately profiled known, variant, and novel microRNAs expressed by endothelial cells in normoxia and hypoxia.

Pseudomonas aeruginosa: Infections and novel approaches to treatment "Knowing the enemy" the threat of Pseudomonas aeruginosa and exploring novel approaches to treatment.

Pseudomonas aeruginosa is an aerobic Gram-negative rod-shaped bacterium with a comparatively large genome and an impressive genetic capability allowing it to grow in a variety of environments and tolerate a wide range of physical conditions. This biological flexibility enables the P. aeruginosa to cause a broad range of infections in patients with serious underlying medical conditions, and to be a principal cause of health care associated infection worldwide. The clinical manifestations of P. aeruginosa include mostly health care associated infections and community-acquired infections. P. aeruginosa possesses an array of virulence factors that counteract host defence mechanisms. It can directly damage host tissue while utilizing high levels of intrinsic and acquired antimicrobial resistance mechanisms to counter most classes of antibiotics. P. aeruginosa co-regulates multiple resistance mechanisms by perpetually moving targets poses a significant therapeutic challenge. Thus, there is an urgent need for novel approaches in the development of anti-Pseudomonas agents. Here we review the principal infections caused by P. aeruginosa and we discuss novel therapeutic options to tackle antibiotic resistance and treatment of P. aeruginosa infections that may be further developed for clinical practice.

Insights into the Evolution of P. aeruginosa Antimicrobial Resistance in a Patient Undergoing Intensive Therapy.

Whole genome sequencing (WGS) provides insights into the evolution of antimicrobial resistance, an urgent global health threat. Using WGS, we observe evolutionary adaptation of a Pseudomonas aeruginosa strain within an immunocompromised patient undergoing antibiotic therapy. Two blood isolates (EA-86 and EA-87) from the patient evolved separate adaptations for antibiotic resistance, while sharing common adaptive mutations for host immune evasion. In EA-86, a silencing mutation in the antibiotic efflux pump repressor, NfxB, increased antibiotic resistance, while in EA-87, a similar mutation was seen in the antibiotic efflux pump repressor mexR. The number of genomic variants between the two isolates give a divergence time estimate of the order of 1000 generations. This time is sufficient for a bacterial lineage to have evolved an SNP in every position in the genome and been fixed if advantageous. This demonstrates the evolutionary adaptive power accessible to bacteria and the timescale for a brute-force functional survey of the SNP fitness landscape.

The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Intron splicing suppresses RNA silencing in Arabidopsis.

Silencing of introduced transgenes constitutes a major bottleneck in the production of transgenic crops. Commonly, these transgenes contain no introns, a feature shared with transposons, which are also prime targets for gene silencing. Given that introns are very common in endogenous genes but are often lacking in transgenes and transposons, we hypothesised that introns may suppress gene silencing. To investigate this, we conducted a genome-wide analysis of small RNA densities in exons from intronless versus intron-containing genes in Arabidopsis thaliana. We found that small RNA libraries are strongly enriched for exon sequences derived from intronless genes. Small RNA densities in exons of intronless genes were comparable to exons of transposable elements. To test these findings in vivo we used a transgenic reporter system to determine whether introns are able to suppress gene silencing in Arabidopsis. Introducing an intron into a transgene reduced silencing by more than fourfold. Compared with intronless transcripts, the spliced transcripts were less effective substrates for RNA-dependent RNA polymerase 6-mediated gene silencing. This intron suppression of transgene silencing requires efficient intron splicing and is dependent on ABH1, the Arabidopsis orthologue of human cap-binding protein 80.

Methylomic and transcriptomic characterization of postoperative systemic inflammatory dysregulation.

In this study, we define and validate a state of postoperative systemic inflammatory dysregulation (PSID) based on postoperative phenotypic extremes of plasma C-reactive protein concentration following major abdominal surgery. PSID manifested clinically with significantly higher rates of sepsis, complications, longer hospital stays and poorer short, and long-term outcomes. We hypothesized that PSID will be associated with, and potentially predicted by, altered patterns of genome-wide peripheral blood mononuclear cell differential DNA methylation and gene expression. We identified altered DNA methylation and differential gene expression in specific immune and metabolic pathways during PSID. Our findings suggest that dysregulation results in, or from, dramatic changes in differential DNA methylation and highlights potential targets for early detection and treatment. The combination of altered DNA methylation and gene expression suggests that dysregulation is mediated at multiple levels within specific gene sets and hence, nonspecific anti-inflammatory treatments such as corticosteroids alone are unlikely to represent an effective therapeutic strategy.

Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression.

Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.

Evaluation of polymorphisms in predicted target sites for micro RNAs differentially expressed in endometriosis.

Previous microarray analyses identified 22 microRNAs (miRNAs) differentially expressed in paired ectopic and eutopic endometrium of women with and without endometriosis. To investigate further the role of these miRNAs in women with endometriosis, we conducted an association study aiming to explore the relationship between endometriosis risk and single-nucleotide polymorphisms (SNPs) in miRNA target sites for these differentially expressed miRNAs. A panel of 102 SNPs in the predicted miRNA binding sites were evaluated for an endometriosis association study and an ingenuity pathway analysis was performed. Fourteen rare variants were identified in this study. We found SNP rs14647 in the Wolf-Hirschhorn syndrome candidate gene1 (WHSC1) 3'UTR (untranslated region) was associated with endometriosis-related infertility presenting an odds ratio of 12.2 (95% confidence interval = 2.4-60.7, P = 9.03 × 10(-5)). SNP haplotype AGG in the solute carrier family 22, member 23 (SLC22A23) 3'UTR was associated with endometriosis-related infertility and more severe disease. With the individual genotyping data, ingenuity pathways analysis identified the tumour necrosis factor and cyclin-dependant kinase inhibitor as major factors in the molecular pathways. Significant associations between WHSC1 alleles and endometriosis-related infertility and SLC22A23 haplotypes and the disease severe stage were identified. These findings may help focus future research on subphenotypes of this disease. Replication studies in independent large sample sets to confirm and characterize the involvement of the gene variation in the pathogenesis of endometriosis are needed.

MicroRNAs in the shoot apical meristem of soybean.

Plant microRNAs (miRNAs) play crucial regulatory roles in various developmental processes. In this study, we characterize the miRNA profile of the shoot apical meristem (SAM) of an important legume crop, soybean, by integrating high-throughput sequencing data with miRNA microarray analysis. A total of 8423 non-redundant sRNAs were obtained from two libraries derived from micro-dissected SAM or mature leaf tissue. Sequence analysis allowed the identification of 32 conserved miRNA families as well as 8 putative novel miRNAs. Subsequent miRNA profiling with microarrays verified the expression of the majority of these conserved and novel miRNAs. It is noteworthy that several miRNAs* were expressed at a level similar to or higher than their corresponding mature miRNAs in SAM or mature leaf, suggesting a possible biological function for the star species. In situ hybridization analysis revealed a distinct spatial localization pattern for a conserved miRNA, miR166, and its star speciessuggesting that they serve different roles in regulating leaf development. Furthermore, localization studies showed that a novel soybean miRNA, miR4422a, was nuclear-localized. This study also indicated a novel expression pattern of miR390 in soybean. Our approach identified potential key regulators and provided vital spatial information towards understanding the regulatory circuits in the SAM of soybean during shoot development.

Discovery Genome-Wide Association Study of Body Composition in 4,386 Adults From the UK Biobank's Pilot Imaging Enhancement Study.

Body composition (fat, skeletal muscle and bone mass) is an important determinant of overall health and risk of endocrine disorders such as type 2 diabetes and osteoporosis. Although diet and physical activity are strongly implicated, body composition is also heritable. We conducted a discovery genome-wide association study on 31 phenotypes from the three-compartment body composition model (fat, lean and bone mass) in a set of 4 386 individuals (n = 2 109 males, n = 2 294 females) from the UK Biobank pilot imaging enhancement program that underwent a dual energy X-ray absorptiometry (DXA) scan for assessment of body composition and genetic screening. From 6 137 607 imputed single nucleotide polymorphisms (SNPs) we identified 17 body composition loci (P<5.0 x 10-8). GWAS from the combined dataset identified four statistically significant SNPs (rs7592270, rs145972737, rs13212044, rs77772562). In sex-stratified GWAS, 10 male specific SNPs across all traits were identified and five female specific SNPs. Of the 17 SNPs, six were in or close to a gene where there was a plausible functional connection. Three SNPs (rs7592270, rs77772562 and rs7552312) were correlated with obesity phenotypes, one SNP (rs2236705) with lean phenotypes and two with bone mass phenotypes (rs112098641 and rs113380185). These results highlight candidate genes and biological pathways related to body composition, including glucose metabolism and estrogen regulation, that are of interest to replicate in future studies.

Dataset for genome sequencing and de novo assembly of the Vietnamese bighead catfish (Clarias macrocephalus Günther, 1864).

Freshwater catfish of the genus Clarias, known as the airbreathing catfish, are widespread and important for food security through small scale inland fisheries and aquaculture. Limited genomic data are available for this important group of fishes. The bighead catfish (Clarias macrocephalus) is a commercial aquaculture species in southeast Asia used for aquaculture and threatened in its natural environment through habitat destruction, over-exploitation and competition from other introduced species of Clarias. Despite its commercial importance and threats to natural populations, public databases do not include any genomic data for C. macrocephalus. We present the first genomic data for the bighead catfish from Illumina sequencing. A total of 128 Gb of sequence data in paired-end 150 bp reads were assembled de novo, generating a final assembly of 883 Mbp contained in 27,833 scaffolds (N50 length: 80.8 kbp) with BUSCO completeness assessments of 96.3% and 87.6% based on metazoan and Actinopterygii ortholog datasets, respectively. Annotation of the genome predicted 21,124 gene sequences, which were assigned putative functions based on homology to existing protein sequences in public databases. Raw fastq reads and the final version of the genome assembly have been deposited in the NCBI (BioProject: PRJNA604477, WGS: JAAGKR000000000, SRA: SRR11188453). The complete C. macrocephalus mitochondrial genome was also recovered from the same sequence read dataset and is available on NCBI (accession: MT109097), representing the first mitogenome for this species. Lastly, we find an expansion of the mb and ora1 genes thought to be associated with adaptations to air-breathing and a semi-terrestrial life style in this genus of catfish.

A transcriptional sketch of a primary human breast cancer by 454 deep sequencing.

BACKGROUND: The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. RESULTS: We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. CONCLUSION: Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling.

Chromosome-Level Alpaca Reference Genome VicPac3.1 Improves Genomic Insight Into the Biology of New World Camelids.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago® chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly VicPac3.1 contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the Minute Chromosome Syndrome (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome VicPac3.1 presents a significant improvement in completeness, contiguity and accuracy over VicPac2 and is an important tool for the advancement of genomics research in all New World camelids.

Gender-Specific Molecular and Clinical Features Underlie Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (P < 0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that nonepitheliod histology (P = 0.037), whereas CDKN2A deletions occurred more frequently in nonepithelioid subtypes among men (P = 0.021) and were correlated with shorter overall survival for the entire cohort (P = 0.002) and for men (P = 0.012). Furthermore, women were more likely to harbor TP53 mutations (P = 0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in nonepithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM.

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

BACKGROUND: Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. RESULTS: TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. CONCLUSIONS: Amresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.

Whole Genome Sequencing of the Asian Arowana (Scleropages formosus) Provides Insights into the Evolution of Ray-Finned Fishes.

The Asian arowana (Scleropages formosus) is of commercial importance, conservation concern, and is a representative of one of the oldest lineages of ray-finned fish, the Osteoglossomorpha. To add to genomic knowledge of this species and the evolution of teleosts, the genome of a Malaysian specimen of arowana was sequenced. A draft genome is presented consisting of 42,110 scaffolds with a total size of 708 Mb (2.85% gaps) representing 93.95% of core eukaryotic genes. Using a k-mer-based method, a genome size of 900 Mb was also estimated. We present an update on the phylogenomics of fishes based on a total of 27 species (23 fish species and 4 tetrapods) using 177 orthologous proteins (71,360 amino acid sites), which supports established relationships except that arowana is placed as the sister lineage to all teleost clades (Bayesian posterior probability 1.00, bootstrap replicate 93%), that evolved after the teleost genome duplication event rather than the eels (Elopomorpha). Evolutionary rates are highly heterogeneous across the tree with fishes represented by both slowly and rapidly evolving lineages. A total of 94 putative pigment genes were identified, providing the impetus for development of molecular markers associated with the spectacular colored phenotypes found within this species.

Multiple independent analyses reveal only transcription factors as an enriched functional class associated with microRNAs.

BACKGROUND: Transcription factors (TFs) have long been known to be principally activators of transcription in eukaryotes and prokaryotes. The growing awareness of the ubiquity of microRNAs (miRNAs) as suppressive regulators in eukaryotes, suggests the possibility of a mutual, preferential, self-regulatory connectivity between miRNAs and TFs. Here we investigate the connectivity from TFs and miRNAs to other genes and each other using text mining, TF promoter binding site and 6 different miRNA binding site prediction methods. RESULTS: In the first approach text mining of PubMed abstracts reveal statistically significant associations between miRNAs and both TFs and signal transduction gene classes. Secondly, prediction of miRNA targets in human and mouse 3'UTRs show enrichment only for TFs but not consistently across prediction methods for signal transduction or other gene classes. Furthermore, a random sample of 986 TarBase entries was scored for experimental evidence by manual inspection of the original papers, and enrichment for TFs was observed to increase with score. Low-scoring TarBase entries, where experimental evidence is anticorrelated miRNA:mRNA expression with predicted miRNA targets, appear not to select for real miRNA targets to any degree. Our manually validated text-mining results also suggests that miRNAs may be activated by more TFs than other classes of genes, as 7% of miRNA:TF co-occurrences in the literature were TFs activating miRNAs. This was confirmed when thirdly, we found enrichment for predicted, conserved TF binding sites in miRNA and TF genes compared to other gene classes. CONCLUSIONS: We see enrichment of connections between miRNAs and TFs using several independent methods, suggestive of a network of mutual activating and suppressive regulation. We have also built regulatory networks (containing 2- and 3-loop motifs) for mouse and human using predicted miRNA and TF binding sites and we have developed a web server to search and display these loops, available for the community at http://rth.dk/resources/tfmirloop.