Optimal cDNA microarray design using expressed sequence tags for organisms with limited genomic information

BACKGROUND: Expression microarrays are increasingly used to characterize environmental responses and host-parasite interactions for many different organisms. Probe selection for cDNA microarrays using expressed sequence tags (ESTs) is challenging due to high sequence redundancy and potential cross-hybridization between paralogous genes. In organisms with limited genomic information, like marine organisms, this challenge is even greater due to annotation uncertainty. No general tool is available for cDNA microarray probe selection for these organisms. Therefore, the goal of the design procedure described here is to select a subset of ESTs that will minimize sequence redundancy and characterize potential cross-hybridization while providing functionally representative probes. RESULTS: Sequence similarity between ESTs, quantified by the E-value of pair-wise alignment, was used as a surrogate for expected hybridization between corresponding sequences. Using this value as a measure of dissimilarity, sequence redundancy reduction was performed by hierarchical cluster analyses. The choice of how many microarray probes to retain was made based on an index developed for this research: a sequence diversity index (SDI) within a sequence diversity plot (SDP). This index tracked the decreasing within-cluster sequence diversity as the number of clusters increased. For a given stage in the agglomeration procedure, the EST having the highest similarity to all the other sequences within each cluster, the centroid EST, was selected as a microarray probe. A small dataset of ESTs from Atlantic white shrimp (Litopenaeus setiferus) was used to test this algorithm so that the detailed results could be examined. The functional representative level of the selected probes was quantified using Gene Ontology (GO) annotations. CONCLUSIONS: For organisms with limited genomic information, combining hierarchical clustering methods to analyze ESTs can yield an optimal cDNA microarray design. If biomarker discovery is the goal of the microarray experiments, the average linkage method is more effective, while single linkage is more suitable if identification of physiological mechanisms is more of interest. This general design procedure is not limited to designing single-species cDNA microarrays for marine organisms, and it can equally be applied to multiple-species microarrays of any organisms with limited genomic information

Marine Genomics: A clearing-house for genomic and transcriptomic data of marine organisms

BACKGROUND: The Marine Genomics project is a functional genomics initiative developed to provide a pipeline for the curation of Expressed Sequence Tags (ESTs) and gene expression microarray data for marine organisms. It provides a unique clearing-house for marine specific EST and microarray data and is currently available at . DESCRIPTION: The Marine Genomics pipeline automates the processing, maintenance, storage and analysis of EST and microarray data for an increasing number of marine species. It currently contains 19 species databases (over 46,000 EST sequences) that are maintained by registered users from local and remote locations in Europe and South America in addition to the USA. A collection of analysis tools are implemented. These include a pipeline upload tool for EST FASTA file, sequence trace file and microarray data, an annotative text search, automated sequence trimming, sequence quality control (QA/QC) editing, sequence BLAST capabilities and a tool for interactive submission to GenBank. Another feature of this resource is the integration with a scientific computing analysis environment implemented by MATLAB. CONCLUSION: The conglomeration of multiple marine organisms with integrated analysis tools enables users to focus on the comprehensive descriptions of transcriptomic responses to typical marine stresses. This cross species data comparison and integration enables users to contain their research within a marine-oriented data management and analysis environment

Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)-dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1)

Force generation in smooth muscle is driven by phosphorylation of myosin light chains (MYL), which is regulated by the equilibrium between the activities of myosin light chain kinase (MYLK) and myosin phosphatase (MYLP). MYLK is activated by Ca2+-calmodulin whereas MYLP is inhibited by phosphorylation of its myosin-binding subunit (MYPT1) by Ca2+-independent mechanisms, leading to generation of increased MYL phosphorylation and force for a given intracellular Ca2+ concentration, a phenomenon known as ‘calcium-sensitization’. The regulation of MYPT1 phosphorylation in human myometrium, which shows increasing phasic contractility at the onset of labour, has yet to be fully investigated. Here, we explore phosphorylation of MYPT1 at Thr696 and Thr853, alongside phosphorylation of MYL, in fresh human myometrial tissue and cultured myometrial cells. We report that pMYPT1 (Thr853) levels are dependent on the activity of Rho-associated kinase (ROCK), determined using the ROCK inhibitor g-H-1152 and siRNA-mediated knockdown of ROCK1/2, and are highly correlated to ppMYL (Thr18/Ser19) levels. Pharmacological inhibition of ROCK was associated with a decrease in oxytocin (OXT)-stimulated contractility of myometrial strips in vitro. Moreover, we have measured pMYPT1 and pMYL levels between and during spontaneous and OXT-stimulated phasic contractions by rapidly freezing contracting muscle, and demonstrate for the first time functional coupling between increases in pMYPT1 (Thr853), ppMYL (Thr18/Ser19) and phasic contractility that is ROCK-dependent. The combined approach of measuring contractility and phosphorylation has demonstrated that the phosphorylation of MYPT1 (Thr853) changes dynamically with each contraction and has elucidated a defined role for ROCK in regulating myometrial contractility through MYLP, providing new insights into uterine physiology which will stimulate further research into treatments for preterm labour

Investigating porcine parvoviruses genogroup 2 infection using in situ polymerase chain reaction

Abstract Background Porcine parvovirus 2 (PPV2) was detected in swine serum without showing any relationship with disease. The emergence of the virus seemed to be a unique event until other genetically highly similar parvoviruses were identified in China and, later in 2012, the presence of the virus was also described in Europe. PPV2 is widely distributed in pig populations where it is suspected to be involved in respiratory conditions, based on its frequent detection in lung samples. In order to investigate the potential pathogenic involvement of PPV2, 60 dead pigs were examined from two farms. They were necropsied and tested for PPV2 and PCV2 (Porcine circovirus type 2) by PCR; by Brown and Brenn (B&B) staining for bacteria; by immunohistochemistry (IHC) to detect CD3, Swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza (SIV), Mycoplasma hyopneumoniae (Mhyo); and by in situ hybridization (ISH) to detect ssDNA and dsDNA of PCV2. PPV2 positive samples were subjected to in situ polymerase chain reaction (IS-PCR) including double staining method to detect PPV2 and host cell markers. To calculate statistical difference we used GENMOD or LOGISTIC procedures in Statistical Analysis System (SAS®). Results We found that the PPV2 was localized mostly in lymphocytes in lungs, lymph nodes and liver. Neither CD3 antigen nor lysozyme was expressed by these infected cells. In contrast, low levels of SLAIIDQ were expressed by infected cells, suggesting that PPV2 may have a specific tropism for immature B lymphocytes and/or NK lymphocytes though possibly not T lymphocytes. Conclusion The overall conclusion of this study indicates that PPV2 may contribute to the pathogenesis of pneumonia

Histone H2A (H2A.X and H2A.Z) Variants in Molluscs: Molecular Characterization and Potential Implications For Chromatin Dynamics

Histone variants are used by the cell to build specialized nucleosomes, replacing canonical histones and generating functionally specialized chromatin domains. Among many other processes, the specialization imparted by histone H2A (H2A.X and H2A.Z) variants to the nucleosome core particle constitutes the earliest response to DNA damage in the cell. Consequently, chromatin-based genotoxicity tests have been developed in those cases where enough information pertaining chromatin structure and dynamics is available (i.e., human and mouse). However, detailed chromatin knowledge is almost absent in most organisms, specially protostome animals. Molluscs (which represent sentinel organisms for the study of pollution) are not an exception to this lack of knowledge. In the present work we first identified the existence of functionally differentiated histone H2A.X and H2A.Z variants in the mussel Mytilus galloprovincialis (MgH2A.X and MgH2A.Z), a marine organism widely used in biomonitoring programs. Our results support the functional specialization of these variants based on: a) their active expression in different tissues, as revealed by the isolation of native MgH2A.X and MgH2A.Z proteins in gonad and hepatopancreas; b) the evolutionary conservation of different residues encompassing functional relevance; and c) their ability to confer specialization to nucleosomes, as revealed by nucleosome reconstitution experiments using recombinant MgH2A.X and MgH2A.Z histones. Given the seminal role of these variants in maintaining genomic integrity and regulating gene expression, their preliminary characterization opens up new potential applications for the future development of chromatin-based genotoxicity tests in pollution biomonitoring programs

Development and evaluation of novel molecular assays for the detection of economically important viruses for farm animals

In this project a number of novel molecular assays were developed and evaluated as potential tools for detection, genotyping and quantifying economically important viruses of farm animals. Molecular Beacon assays were developed for an African swine fever Virus, Aujeszky's disease virus, porcine circovirus type 2 and porcine parvovirus. The technology was found to be capable of the rapid, sensitive and specific detection of viral nucleic acids to an equivalent or better standard than the conventional peR assays used in our laboratories. 5'-conjugated minor groove binder assays were applied to the detection of African swine fever virus, porcine circovirus type 2, and porcine parvovirus, swine vesicular disease virus and foot and mouth disease. The performance of these assays was equivalent to those of published assays and, in the case of foot and mouth disease virus and swine vesicular disease virus, to the assays currently used in the UK reference laboratories. A 5'-conjugated minor groove binder assay and a Primer Probe Energy Transfer (PriProET) assay were developed for the differential detection, and potential genotyping of avian infectious laryngotracheitis virus. These assays were compared to TaqMan and conventional peR assays. The 5'-conjugated minor groove binder assay did not perform as well as the TaqMan assay in terms of diagnostic sensitivy. The PriProET assay proved to be equivalent to the TaqMan assay. Both assays were capable of detecting single base changes in the viral target and could therefore potentially function as genotyping assays. The porcine circovirus type 2 5'-conjugated minor groove binder assay was applied to the quantification of viral load in a vaccine study. The assay was capable of quantifying viral load in vaccinated and non-vaccinated groups of animals, in tissues, sera and faeces. The assay performed better in terms of efficiency than a SYBR Green I assay but would require further validation in order to establish the accuracy of quantification, particular if it was to be used for diagnosis of "histopatological" post-weaning multisystemic wasting syndrome. A loop-mediated isothermal amplification assay was applied for the detection of avian infectious laryngotracheitis virus. The assay had 1 Q-fold reduced analytical sensitivity when compared-to real-time peR assays but showed good diagnostic sensitivity. Initial evaluation of the use of Eva Green for possible pen-side detection was promising.EThOS - Electronic Theses Online ServiceGBUnited Kingdo