To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

Fungi (Ascomycota and Basidiomycota) are prolific producers of structurally diverse terpenoid compounds. Classes of terpenoids identified in fungi include the sesqui-, di- and triterpenoids. Biosynthetic pathways and enzymes to terpenoids from each of these classes have been described. These typically involve the scaffold generating terpene synthases and cyclases, and scaffold tailoring enzymes such as e.g. cytochrome P450 monoxygenases, NAD(P)+ and flavin dependent oxidoreductases, and various group transferases that generate the final bioactive structures. The biosynthesis of several sesquiterpenoid mycotoxins and bioactive diterpenoids has been well-studied in Ascomycota (e.g. filamentous fungi). Little is known about the terpenoid biosynthetic pathways in Basidiomycota (e.g. mushroom forming fungi), although they produce a huge diversity of terpenoid natural products. Specifically, many trans-humulyl cation derived sesquiterpenoid natural products with potent bioactivities have been isolated. Biosynthetic gene clusters responsible for the production of trans-humulyl cation derived protoilludanes, and other sesquiterpenoids, can be rapidly identified by genome sequencing and bioinformatic methods. Genome mining combined with heterologous biosynthetic pathway refactoring has the potential to facilitate discovery and production of pharmaceutically relevant fungal terpenoids.

Basidiomycota represent a diverse source of natural products, particularly the sesquiterpenoids. Recently, the genome sequencing, mining, and subsequent discovery of a suite of sesquiterpene synthases was described in Omphalotus olearius. A predictive framework was developed to facilitate the discovery of sesquiterpene synthases in Basidiomycota. Phylogenetic analyses indicated a conservation of both sequence and initial cyclization mechanisms used. Here, the first robust application of this predictive framework is reported. It is used to pursue and selectively identify sesquiterpene synthases that follow a 1,6-, 1,10-, and 1,11-cyclization mechanism in the crust fungus Stereum hirsutum. The successful identification and characterization of a 1,6- and a 1,10-cyclizing sesquiterpene synthase, as well as three 1,11-cyclizing Δ-6 protoilludene synthases, is described. This study verifies the accuracy and utility of the predictive framework as a roadmap for the discovery of specific sesquiterpene synthases from Basidiomycota, representing an important step forward in natural product discovery.

We describe SPINN (Straightforward Pulsar Identification using Neural Networks), a high-performance machine learning solution developed to process increasingly large data outputs from pulsar surveys. SPINN has been cross-validated on candidates from the southern High Time Resolution Universe (HTRU) survey and shown to identify every known pulsar found in the survey data while maintaining a false positive rate of 0.64%. Furthermore, it ranks 99% of pulsars among the top 0.11% of candidates, and 95% among the top 0.01%. In conjunction with the peasoup pipeline (Barr et al. in preparation), it has already discovered four new pulsars in a re-processing of the intermediate Galactic latitude area of HTRU, three of which have spin periods shorter than 5 milliseconds. SPINN's ability to reduce the amount of candidates to visually inspect by up to four orders of magnitude makes it a very promising tool for future large-scale pulsar surveys. In an effort to provide a common testing ground for pulsar candidate selection tools and stimulate interest in their development, we also make publicly available the set of candidates on which SPINN was cross-validated.

We present initial results from the low-latitude Galactic plane region of the High Time Resolution Universe pulsar survey conducted at the Parkes 64-m radio telescope. We discuss the computational challenges arising from the processing of the terabyte-sized survey data. Two new radio interference mitigation techniques are introduced, as well as a partially-coherent segmented acceleration search algorithm which aims to increase our chances of discovering highly-relativistic short-orbit binary systems, covering a parameter space including potential pulsar-black hole binaries. We show that under a constant acceleration approximation, a ratio of data length over orbital period of ≈ 0.1 results in the highest effectiveness for this search algorithm. From the 50 per cent of data processed thus far, we have re-detected 435 previously known pulsars and discovered a further 60 pulsars, two of which are fast-spinning pulsars with periods less than 30 ms. PSR J1101−6424 is a millisecond pulsar whose heavy white dwarf (WD) companion and short spin period of 5.1 ms indicate a rare example of full-recycling via Case A Roche lobe overflow. PSR J1757−27 appears to be an isolated recycled pulsar with a relatively long spin period of 17 ms. In addition, PSR J1244−6359 is a mildly-recycled binary system with a heavy WD companion, PSR J1755−25 has a significant orbital eccentricity of 0.09, and PSR J1759−24 is likely to be a long-orbit eclipsing binary with orbital period of the order of tens of years. Comparison of our newly-discovered pulsar sample to the known population suggests that they belong to an older population. Furthermore, we demonstrate that our current pulsar detection yield is as expected from population synthesis.

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.

To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, ␤-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low-and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of ␤-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high-and low-risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.

Cellular pools of free arachidonic acid are tightly controlled through enzymatic release of the fatty acid and subsequent utilization by downstream enzymes including the cyclooxygenases. Arachidonic acid cleavage from membrane phospholipids is accomplished by the actions of phospholipase A 2 (PLA 2 ). Upon release, free arachidonic acid provides substrate for the synthesis of eicosanoids. However, under certain conditions, arachidonic acid may participate in ceramidemediated apoptosis. Disruption of arachidonic acid homeostasis can shift the balance of cell turnover in favor of tumorigenesis, via overproduction of tumor-promoting eicosanoids or alternatively by limiting proapoptotic signals. In the following study, we evaluated the influence of genetic deletion of a key intracellular phospholipase, cytoplasmic PLA 2 (cPLA 2 ), on azoxymethane-induced colon tumorigenesis. Heterozygous and null mice, upon treatment with the organotropic colon carcinogen, azoxymethane, developed a significant (P < 0.05) increase in colon tumor multiplicity (7.2-fold and 5.5-fold, respectively) relative to their wild-type littermates. This enhanced tumor sensitivity may be explained, in part, by the attenuated levels of apoptosis observed by terminal deoxynucleotidyl transferase-mediated nick end labeling staining within the colonic epithelium of heterozygous and null mice (% % %50% of wild type). The lower frequency of apoptotic cells corresponded with reduced ceramide levels (69% and 46% of wild-type littermates, respectively). Remarkably, increased tumorigenesis resulting from cPLA 2 deletion occurred despite a significant reduction in prostaglandin E 2 production, even in cyclooxygenase-2-overexpressing tumors. These data contribute new information that supports a fundamental role of cPLA 2 in the control of arachidonic acid homeostasis and cell turnover. Our findings indicate that the proapoptotic role of cPLA 2 in the colon may supercede its contribution to eicosanoid production in tumor development.