Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. <p/>Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. <p/>Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. <p/>Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters

Regulation of S1PR2 by the EBV oncogene LMP1 in aggressive ABC subtype diffuse large B cell lymphoma.

The Epstein-Barr virus (EBV) is found almost exclusively in the activated B cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL), yet its contribution to this tumour remains poorly understood. We have focussed on the EBV-encoded latent membrane protein-1 (LMP1), a constitutively activated CD40 homologue expressed in almost all EBV-positive DLBCL and which can disrupt germinal centre (GC) formation and drive lymphomagenesis in mice. Comparison of the transcriptional changes that follow LMP1 expression with those that follow transient CD40 signalling in human GC B cells enabled us to define pathogenic targets of LMP1 aberrantly expressed in ABC-DLBCL. These included the down-regulation of S1PR2, a sphingosine-1-phosphate (S1P) receptor that is transcriptionally down-regulated in ABC-DLBCL, and when genetically ablated leads to DLBCL in mice. Consistent with this we found that LMP1-expressing primary ABC-DLBCL were significantly more likely to lack S1PR2 expression than were LMP1-negative tumours. Furthermore, we showed that the down-regulation of S1PR2 by LMP1 drives a signalling loop leading to constitutive activation of the phosphatidylinositol-3-kinase (PI3-K) pathway. Finally, core LMP1-PI3-K targets were enriched for lymphoma-related transcription factors and genes associated with shorter overall survival in patients with ABC-DLBCL. Our data identify a novel function for LMP1 in aggressive DLBCL

Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation

The aim of this study was to apply a generated Δtku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δtku70Δlcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1

GAIA: a gram-based interaction analysis tool – an approach for identifying interacting domains in yeast

<p>Abstract</p> <p>Background</p> <p>Protein-Protein Interactions (PPIs) play important roles in many biological functions. Protein domains, which are defined as independently folding structural blocks of proteins, physically interact with each other to perform these biological functions. Therefore, the identification of Domain-Domain Interactions (DDIs) is of great biological interests because it is generally accepted that PPIs are mediated by DDIs. As a result, much effort has been put on the prediction of domain pair interactions based on computational methods. Many DDI prediction tools using PPIs network and domain evolution information have been reported. However, tools that combine the primary sequences, domain annotations, and structural annotations of proteins have not been evaluated before.</p> <p>Results</p> <p>In this study, we report a novel approach called Gram-bAsed Interaction Analysis (GAIA). GAIA extracts peptide segments that are composed of fixed length of continuous amino acids, called n-grams (where n is the number of amino acids), from the annotated domain and DDI data set in <it>Saccharomyces cerevisiae </it>(budding yeast) and identifies a list of n-grams that may contribute to DDIs and PPIs based on the frequencies of their appearance. GAIA also reports the coordinate position of gram pairs on each interacting domain pair. We demonstrate that our approach improves on other DDI prediction approaches when tested against a gold-standard data set and achieves a true positive rate of 82% and a false positive rate of 21%. We also identify a list of 4-gram pairs that are significantly over-represented in the DDI data set and may mediate PPIs.</p> <p>Conclusion</p> <p>GAIA represents a novel and reliable way to predict DDIs that mediate PPIs. Our results, which show the localizations of interacting grams/hotspots, provide testable hypotheses for experimental validation. Complemented with other prediction methods, this study will allow us to elucidate the interactome of cells.</p

Factors Associated with Bovine Neonatal Pancytopenia (BNP) in Calves: A Case-Control Study

Bovine neonatal pancytopenia (BNP; previously known as idiopathic haemorrhagic diathesis and commonly known as bleeding calf syndrome) is a novel haemorrhagic disease of young calves which has emerged in a number of European countries during recent years. Data were retrospectively collected during June to November 2010 for 56 case calves diagnosed with BNP between 17 March and 7 June of the same year. These were compared with 58 control calves randomly recruited from herds with no history of BNP. Multivariable logistic regression analysis showed that increased odds of a calf being a BNP case were associated with its dam having received PregSure® BVD (Pfizer Animal Health) vaccination prior to the birth of the calf (odds ratio (OR) 40.78, p<0.001) and its herd of origin being located in Scotland (OR 9.71, p = 0.006). Decreased odds of a calf being a BNP case were associated with the calf having been kept outside (OR 0.11, p = 0.006). The longer that a cattle herd had been established on the farm was also associated with decreased odds of a calf in that herd being a BNP case (OR 0.97, p = 0.011)