Genome-wide analysis clarifies the population genetic structure of wild gilthead sea bream (Sparus aurata).

Gilthead sea bream is an important target for both recreational and commercial fishing in Europe, where it is also one of the most important cultured fish. Its distribution ranges from the Mediterranean to the African and European coasts of the North-East Atlantic. Until now, the population genetic structure of this species in the wild has largely been studied using microsatellite DNA markers, with minimal genetic differentiation being detected. In this geographically widespread study, 958 wild gilthead sea bream from 23 locations within the Mediterranean Sea and Atlantic Ocean were genotyped at 1159 genome-wide SNP markers by RAD sequencing. Outlier analyses identified 18 loci potentially under selection. Neutral marker analyses identified weak subdivision into three genetic clusters: Atlantic, West, and East Mediterranean. The latter group could be further subdivided into an Ionian/Adriatic and an Aegean group using the outlier markers alone. Seascape analysis suggested that this differentiation was mainly due to difference in salinity, this being also supported by preliminary genomic functional analysis. These results are of fundamental importance for the development of proper management of this species in the wild and are a first step toward the study of the potential genetic impact of the sea bream aquaculture industry.

Tumor Fusion Burden as a Hallmark of Immune Infiltration in Prostate Cancer.

Prostate cancer is the second leading cause of cancer-related death in men. Despite having a relatively lower tumor mutational burden than most tumor types, multiple gene fusions such as TMPRSS2:ERG have been characterized and linked to more aggressive disease. Individual tumor samples have been found to contain multiple fusions, and it remains unknown whether these fusions increase tumor immunogenicity. Here, we investigated the role of fusion burden on the prevalence and expression of key molecular and immune effectors in prostate cancer tissue specimens that represented the different stages of disease progression and androgen sensitivity, including hormone-sensitive and castration-resistant prostate cancer. We found that tumor fusion burden was inversely correlated with tumor mutational burden and not associated with disease stage. High fusion burden correlated with high immune infiltration, PD-L1 expression on immune cells, and immune signatures, representing activation of T cells and M1 macrophages. High fusion burden inversely correlated with immune-suppressive signatures. Our findings suggest that high tumor fusion burden may be a more appropriate biomarker than tumor mutational burden in prostate cancer, as it more closely associates with immunogenicity, and suggests that tumors with high fusion burden could be potential candidates for immunotherapeutic agents.

Comprehensive Profiling of Poor-Risk Paired Primary and Recurrent Triple-Negative Breast Cancers Reveals Immune Phenotype Shifts.

PURPOSE: Emerging data suggest immune checkpoint inhibitors have reduced efficacy in heavily pretreated triple-negative breast cancers (TNBC), but underlying mechanisms are poorly understood. To better understand the phenotypic evolution of TNBCs, we studied the genomic and transcriptomic profiles of paired tumors from patients with TNBC. EXPERIMENTAL DESIGN: We collected paired primary and metastatic TNBC specimens from 43 patients and performed targeted exome sequencing and whole-transcriptome sequencing. From these efforts, we ascertained somatic mutation profiles, tumor mutational burden (TMB), TNBC molecular subtypes, and immune-related gene expression patterns. Stromal tumor-infiltrating lymphocytes (stromal TIL), recurrence-free survival, and overall survival were also analyzed. RESULTS: We observed a typical TNBC mutational landscape with minimal shifts in copy number or TMB over time. However, there were notable TNBC molecular subtype shifts, including increases in the Lehmann/Pietenpol-defined basal-like 1 (BL1, 11.4%-22.6%) and mesenchymal (M, 11.4%-22.6%) phenotypes, and a decrease in the immunomodulatory phenotype (IM, 31.4%-3.2%). The Burstein-defined basal-like immune-activated phenotype was also decreased (BLIA, 42.2%-17.2%). Among downregulated genes from metastases, we saw enrichment of immune-related Kyoto Encyclopedia of Genes and Genomes pathways and gene ontology (GO) terms, and decreased expression of immunomodulatory gene signatures (P < 0.03) and percent stromal TILs (P = 0.03). There was no clear association between stromal TILs and survival. CONCLUSIONS: We observed few mutational shifts, but largely consistent transcriptomic shifts in longitudinally paired TNBCs. Transcriptomic and IHC analyses revealed significantly reduced immune-activating gene expression signatures and TILs in recurrent TNBCs. These data may explain the observed lack of efficacy of immunotherapeutic agents in heavily pretreated TNBCs. Further studies are ongoing to better understand these initial observations.See related commentary by Savas and Loi, p. 526.

Parallel evolution and adaptation to environmental factors in a marine flatfish: Implications for fisheries and aquaculture management of the turbot (Scophthalmus maximus).

Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot (Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range (672 individuals; 20 populations) by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double-digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming.

Insights into electrosensory organ development, physiology and evolution from a lateral line-enriched transcriptome.

The anamniote lateral line system, comprising mechanosensory neuromasts and electrosensory ampullary organs, is a useful model for investigating the developmental and evolutionary diversification of different organs and cell types. Zebrafish neuromast development is increasingly well understood, but neither zebrafish nor Xenopus is electroreceptive and our molecular understanding of ampullary organ development is rudimentary. We have used RNA-seq to generate a lateral line-enriched gene-set from late-larval paddlefish (Polyodon spathula). Validation of a subset reveals expression in developing ampullary organs of transcription factor genes critical for hair cell development, and genes essential for glutamate release at hair cell ribbon synapses, suggesting close developmental, physiological and evolutionary links between non-teleost electroreceptors and hair cells. We identify an ampullary organ-specific proneural transcription factor, and candidates for the voltage-sensing L-type Cav channel and rectifying Kv channel predicted from skate (cartilaginous fish) ampullary organ electrophysiology. Overall, our results illuminate ampullary organ development, physiology and evolution.

Dedifferentiation of neurons precedes tumor formation in Lola mutants.

The ability to reprogram differentiated cells into a pluripotent state has revealed that the differentiated state is plastic and reversible. It is evident, therefore, that mechanisms must be in place to maintain cells in a differentiated state. Transcription factors that specify neuronal characteristics have been well studied, but less is known about the mechanisms that prevent neurons from dedifferentiating to a multipotent, stem cell-like state. Here, we identify Lola as a transcription factor that is required to maintain neurons in a differentiated state. We show that Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons. In lola mutants, neurons dedifferentiate, turn on neural stem cell genes, and begin to divide, forming tumors. Thus, neurons rather than stem cells or intermediate progenitors are the tumor-initiating cells in lola mutants.

Identification of microRNAs in the coral Stylophora pistillata.

Coral reefs are major contributors to marine biodiversity. However, they are in rapid decline due to global environmental changes such as rising sea surface temperatures, ocean acidification, and pollution. Genomic and transcriptomic analyses have broadened our understanding of coral biology, but a study of the microRNA (miRNA) repertoire of corals is missing. miRNAs constitute a class of small non-coding RNAs of ∼22 nt in size that play crucial roles in development, metabolism, and stress response in plants and animals alike. In this study, we examined the coral Stylophora pistillata for the presence of miRNAs and the corresponding core protein machinery required for their processing and function. Based on small RNA sequencing, we present evidence for 31 bona fide microRNAs, 5 of which (miR-100, miR-2022, miR-2023, miR-2030, and miR-2036) are conserved in other metazoans. Homologues of Argonaute, Piwi, Dicer, Drosha, Pasha, and HEN1 were identified in the transcriptome of S. pistillata based on strong sequence conservation with known RNAi proteins, with additional support derived from phylogenetic trees. Examination of putative miRNA gene targets indicates potential roles in development, metabolism, immunity, and biomineralisation for several of the microRNAs. Here, we present first evidence of a functional RNAi machinery and five conserved miRNAs in S. pistillata, implying that miRNAs play a role in organismal biology of scleractinian corals. Analysis of predicted miRNA target genes in S. pistillata suggests potential roles of miRNAs in symbiosis and coral calcification. Given the importance of miRNAs in regulating gene expression in other metazoans, further expression analyses of small non-coding RNAs in transcriptional studies of corals should be informative about miRNA-affected processes and pathways.

Integrating microRNA and mRNA expression profiling in Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals.

BACKGROUND: Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous smRNAs and potential gene targets in dinoflagellates, we conducted smRNA and mRNA expression profiling over 9 experimental treatments of cultures from Symbiodinium microadriaticum, a photosynthetic symbiont of scleractinian corals. RESULTS: We identified a set of 21 novel smRNAs that share stringent key features with functional microRNAs from other model organisms. smRNAs were predicted independently over all 9 treatments and their putative gene targets were identified. We found 1,720 animal-like target sites in the 3'UTRs of 12,858 mRNAs and 19 plant-like target sites in 51,917 genes. We assembled a transcriptome of 58,649 genes and determined differentially expressed genes (DEGs) between treatments. Heat stress was found to produce a much larger number of DEGs than other treatments that yielded only few DEGs. Analysis of DEGs also revealed that minicircle-encoded photosynthesis proteins seem to be common targets of transcriptional regulation. Furthermore, we identified the core RNAi protein machinery in Symbiodinium. CONCLUSIONS: Integration of smRNA and mRNA expression profiling identified a variety of processes that could be under microRNA control, e.g. protein modification, signaling, gene expression, and response to DNA damage. Given that Symbiodinium seems to have a paucity of transcription factors and differentially expressed genes, identification and characterization of its smRNA repertoire establishes the possibility of a range of gene regulatory mechanisms in dinoflagellates acting post-transcriptionally.

Biochemical diversification through foreign gene expression in bdelloid rotifers.

Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%-9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex.

InterMine: a flexible data warehouse system for the integration and analysis of heterogeneous biological data.

SUMMARY: InterMine is an open-source data warehouse system that facilitates the building of databases with complex data integration requirements and a need for a fast customizable query facility. Using InterMine, large biological databases can be created from a range of heterogeneous data sources, and the extensible data model allows for easy integration of new data types. The analysis tools include a flexible query builder, genomic region search and a library of 'widgets' performing various statistical analyses. The results can be exported in many commonly used formats. InterMine is a fully extensible framework where developers can add new tools and functionality. Additionally, there is a comprehensive set of web services, for which client libraries are provided in five commonly used programming languages. AVAILABILITY: Freely available from http://www.intermine.org under the LGPL license. CONTACT: g.micklem@gen.cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Multiple functionally divergent and conserved copies of alpha tubulin in bdelloid rotifers.

BACKGROUND: Bdelloid rotifers are microscopic animals that have apparently survived without sex for millions of years and are able to survive desiccation at all life stages through a process called anhydrobiosis. Both of these characteristics are believed to have played a role in shaping several unusual features of bdelloid genomes discovered in recent years. Studies into the impact of asexuality and anhydrobiosis on bdelloid genomes have focused on understanding gene copy number. Here we investigate copy number and sequence divergence in alpha tubulin. Alpha tubulin is conserved and normally present in low copy numbers in animals, but multiplication of alpha tubulin copies has occurred in animals adapted to extreme environments, such as cold-adapted Antarctic fish. Using cloning and sequencing we compared alpha tubulin copy variation in four species of bdelloid rotifers and four species of monogonont rotifers, which are facultatively sexual and cannot survive desiccation as adults. Results were verified using transcriptome data from one bdelloid species, Adineta ricciae. RESULTS: In common with the typical pattern for animals, monogonont rotifers contain either one or two copies of alpha tubulin, but bdelloid species contain between 11 and 13 different copies, distributed across five classes. Approximately half of the copies form a highly conserved group that vary by only 1.1% amino acid pairwise divergence with each other and with the monogonont copies. The other copies have divergent amino acid sequences that evolved significantly faster between classes than within them, relative to synonymous changes, and vary in predicted biochemical properties. Copies of each class were expressed under the laboratory conditions used to construct the transcriptome. CONCLUSIONS: Our findings are consistent with recent evidence that bdelloids are degenerate tetraploids and that functional divergence of ancestral copies of genes has occurred, but show how further duplication events in the ancestor of bdelloids led to proliferation in both conserved and functionally divergent copies of this gene.

modMine: flexible access to modENCODE data.

In an effort to comprehensively characterize the functional elements within the genomes of the important model organisms Drosophila melanogaster and Caenorhabditis elegans, the NHGRI model organism Encyclopaedia of DNA Elements (modENCODE) consortium has generated an enormous library of genomic data along with detailed, structured information on all aspects of the experiments. The modMine database (http://intermine.modencode.org) described here has been built by the modENCODE Data Coordination Center to allow the broader research community to (i) search for and download data sets of interest among the thousands generated by modENCODE; (ii) access the data in an integrated form together with non-modENCODE data sets; and (iii) facilitate fine-grained analysis of the above data. The sophisticated search features are possible because of the collection of extensive experimental metadata by the consortium. Interfaces are provided to allow both biologists and bioinformaticians to exploit these rich modENCODE data sets now available via modMine.

The modENCODE Data Coordination Center: lessons in harvesting comprehensive experimental details.

The model organism Encyclopedia of DNA Elements (modENCODE) project is a National Human Genome Research Institute (NHGRI) initiative designed to characterize the genomes of Drosophila melanogaster and Caenorhabditis elegans. A Data Coordination Center (DCC) was created to collect, store and catalog modENCODE data. An effective DCC must gather, organize and provide all primary, interpreted and analyzed data, and ensure the community is supplied with the knowledge of the experimental conditions, protocols and verification checks used to generate each primary data set. We present here the design principles of the modENCODE DCC, and describe the ramifications of collecting thorough and deep metadata for describing experiments, including the use of a wiki for capturing protocol and reagent information, and the BIR-TAB specification for linking biological samples to experimental results. modENCODE data can be found at http://www.modencode.org.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Systemization of leadership for the systemization health care of health care: the unaddressed issue in health care reform.

The case literature strongly suggests that both in England and in Australia health care reforms have had very little impact in terms of "improved performance". It is in the context of a perceived failure in the implementation of the reforms that an interest has arisen in leadership at the level of individual clinical units (e.g an orthopaedics unit or birth unit), as the possible "fix" for bridging the promise-performance gap. Drawing upon extensive case studies that highlight the problem and context for appropriate forms of leadership, this paper argues that the appropriate discourse, in terms of leadership in health reform, needs to focus upon the issue of authorization. In making this argument, addresses the current conceptions of leadership that have been advanced in the discourse before offering some case study material that is suggestive of why attention should be focused on the issue of authorization. Illustrates how and why the processes of leading, central to implementing reform, cannot be construed as socially disembodied processes. Rather, leading and following are partial and partisan processes whose potential is circumscribed by participants' position-takings and what is authorized in the institutional settings in which they are located Argues that the "following" that clinical unit managers could command was shaped by the sub-cultures and "regulatory ideals" with which staff of each profession are involved In the interests of reform, policy players in health should not be focusing attention solely upon the performative qualities and potential leadership abilities of middle level management, but also on their own performance. They should consider how their actions affect what is authorized institutionally and which sets the scope and limits of the leadership-followership dialectic in clinical settings.

Turning back the clock on neural progenitors.

Drosophila neural progenitor cells, or neuroblasts, alter their transcriptional profile over time to produce different neural cell types. A recent paper by Pearson and Doe shows that older neuroblasts can be reprogrammed to behave like young neuroblasts, and to produce early neural cell types, simply by expressing the transcription factor, Hunchback. The authors show that competence to respond to Hunchback diminishes over time. Manipulating neural progenitors in this way may have important implications for therapeutic uses of neural stem cells.