Resistance to white spot syndrome virus in the European shore crab is associated with suppressed virion trafficking and heightened immune responses.

Introduction: All decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. Methods: Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. Results: We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. Discussion: We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

Transformed Canine and Murine Mesenchymal Stem Cells as a Model for Sarcoma with Complex Genomics.

Sarcomas are rare mesenchymal tumors with a broad histological spectrum, but they can be divided into two groups based on molecular pathology: sarcomas with simple or complex genomics. Tumors with complex genomics can have aneuploidy and copy number gains and losses, which hampers the detection of early, initiating events in tumorigenesis. Often, no benign precursors are known, which is why good models are essential. The mesenchymal stem cell (MSC) is the presumed cell of origin of sarcoma. In this study, MSCs of murine and canine origin are used as a model to identify driver events for sarcomas with complex genomic alterations as they transform spontaneously after long-term culture. All transformed murine but not canine MSCs formed sarcomas after subcutaneous injection in mice. Using whole genome sequencing, spontaneously transformed murine and canine MSCs displayed a complex karyotype with aneuploidy, point mutations, structural variants, inter-chromosomal translocations, and copy number gains and losses. Cross-species analysis revealed that point mutations in Tp53/Trp53 are common in transformed murine and canine MSCs. Murine MSCs with a cre-recombinase induced deletion of exon 2-10 of Trp53 transformed earlier compared to wild-type murine MSCs, confirming the contribution of loss of p53 to spontaneous transformation. Our comparative approach using transformed murine and canine MSCs points to a crucial role for p53 loss in the formation of sarcomas with complex genomics.

A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs.

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 µg/L)/linuron (9 or 45 µg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.

Pharmacology beyond the patient - The environmental risks of human drugs.

BACKGROUND: The presence of pharmaceuticals in the environment is a growing global concern and although environmental risk assessment is required for approval of new drugs in Europe and the USA, the adequacy of the current triggers and the effects-based assessments has been questioned. OBJECTIVE: To provide a comprehensive analysis of all regulatory compliant aquatic ecotoxicity data and evaluate the current triggers and effects-based environmental assessments to facilitate the development of more efficient approaches for pharmaceuticals toxicity testing. METHODS: Publicly-available regulatory compliant ecotoxicity data for drugs targeting human proteins was compiled together with pharmacological information including drug targets, Cmax and lipophilicity. Possible links between these factors and the ecotoxicity data for effects on, growth, mortality and/or reproduction, were evaluated. The environmental risks were then assessed based on a combined analysis of drug toxicity and predicted environmental concentrations based on European patient consumption data. RESULTS: For most (88%) of the of 975 approved small molecule drugs targeting human proteins a complete set of regulatory compliant ecotoxicity data in the public domain was lacking, highlighting the need for both intelligent approaches to prioritize legacy human drugs for a tailored environmental risk assessment and a transparent database that captures environmental data. We show that presence/absence of drug-target orthologues are predictive of susceptible species for the more potent drugs. Drugs that target the endocrine system represent the highest potency and greatest risk. However, for most drugs (>80%) with a full set of ecotoxicity data, risk quotients assuming worst-case exposure assessments were below one in all European countries indicating low environmental risks for the endpoints assessed. CONCLUSION: We believe that the presented analysis can guide improvements to current testing procedures, and provide valuable approaches for prioritising legacy drugs (i.e. those registered before 2006) for further ecotoxicity testing. For drugs where effects of possible concern (e.g. behaviour) are not captured in regulatory tests, additional mechanistic testing may be required to provide the highest confidence for avoiding environmental impacts.

ECOdrug: a database connecting drugs and conservation of their targets across species.

Pharmaceuticals are designed to interact with specific molecular targets in humans and these targets generally have orthologs in other species. This provides opportunities for the drug discovery community to use alternative model species for drug development. It also means, however, there is potential for mode of action related effects in non-target wildlife species as many pharmaceuticals reach the environment through patient use and manufacturing wastes. Acquiring insight in drug target ortholog predictions across species and taxonomic groups has proven difficult because of the lack of an optimal strategy and because necessary information is spread across multiple and diverse sources and platforms. We introduce a new research platform tool, ECOdrug, that reliably connects drugs to their protein targets across divergent species. It harmonizes ortholog predictions from multiple sources via a simple user interface underpinning critical applications for a wide range of studies in pharmacology, ecotoxicology and comparative evolutionary biology. ECOdrug can be used to identify species with drug targets and identify drugs that interact with those targets. As such, it can be applied to support intelligent targeted drug safety testing by ensuring appropriate and relevant species are selected in ecological risk assessments. ECOdrug is freely accessible and available at: http://www.ecodrug.org.

Molecular Mechanisms of White Spot Syndrome Virus Infection and Perspectives on Treatments.

Since its emergence in the 1990s, White Spot Disease (WSD) has had major economic and societal impact in the crustacean aquaculture sector. Over the years shrimp farming alone has experienced billion dollar losses through WSD. The disease is caused by the White Spot Syndrome Virus (WSSV), a large dsDNA virus and the only member of the Nimaviridae family. Susceptibility to WSSV in a wide range of crustacean hosts makes it a major risk factor in the translocation of live animals and in commodity products. Currently there are no effective treatments for this disease. Understanding the molecular basis of disease processes has contributed significantly to the treatment of many human and animal pathogens, and with a similar aim considerable efforts have been directed towards understanding host-pathogen molecular interactions for WSD. Work on the molecular mechanisms of pathogenesis in aquatic crustaceans has been restricted by a lack of sequenced and annotated genomes for host species. Nevertheless, some of the key host-pathogen interactions have been established: between viral envelope proteins and host cell receptors at initiation of infection, involvement of various immune system pathways in response to WSSV, and the roles of various host and virus miRNAs in mitigation or progression of disease. Despite these advances, many fundamental knowledge gaps remain; for example, the roles of the majority of WSSV proteins are still unknown. In this review we assess current knowledge of how WSSV infects and replicates in its host, and critique strategies for WSD treatment.

De novo assembly of the Carcinus maenas transcriptome and characterization of innate immune system pathways.

BACKGROUND: The European shore crab, Carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. It is also an important invasive species in numerous global locations. However, the genomic resources for this organism are still sparse, limiting research progress in these fields. To address this resource shortfall we produced a C. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species. RESULTS: We isolated and pooled RNA for twelve different tissues and organs from C. maenas individuals and sequenced the RNA using next generation sequencing on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome was generated encompassing 212,427 transcripts (153,699 loci). The transcripts were filtered, annotated and characterised using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including NCBI, Gene Ontology and KEGG). There were differential patterns of expression for between 1,223 and 2,741 transcripts across tissues and organs with over-represented Gene Ontology terms relating to their specific function. Based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the RNAi, Toll-like receptor signalling, IMD and JAK/STAT pathways. CONCLUSIONS: We have produced an assembled transcriptome for C. maenas that provides a significant molecular resource for wide ranging studies in this species. Analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns.

Integrating protein-protein interaction networks with gene-gene co-expression networks improves gene signatures for classifying breast cancer metastasis.

Multiple studies have illustrated that gene expression profiling of primary breast cancers throughout the final stages of tumor development can provide valuable markers for risk prediction of metastasis and disease sub typing. However, the identification of a biologically interpretable and universally shared set of markers proved to be difficult. Here, we propose a method for de novo grouping of genes by dissecting the protein-protein interaction network into disjoint sub networks using pair wise gene expression correlation measures. We show that the obtained sub networks are functionally coherent and are consistently identified when applied on a compendium composed of six different breast cancer studies. Application of the proposed method using different integration approaches underlines the robustness of the identified sub network related to cell cycle and identifies putative new sub network markers for metastasis related to cell-cell adhesion, the proteasome complex and JUN-FOS signalling. Although gene selection with the proposed method does not directly improve upon previously reported cross study classification performances, it shows great promises for applications in data integration and result interpretation.