ClusTrack: Feature Extraction and Similarity Measures for Clustering of Genome-Wide Data Sets

Clustering is a popular technique for explorative analysis of data, as it can reveal subgroupings and similarities between data in an unsupervised manner. While clustering is routinely applied to gene expression data, there is a lack of appropriate general methodology for clustering of sequence-level genomic and epigenomic data, e.g. ChIP-based data. We here introduce a general methodology for clustering data sets of coordinates relative to a genome assembly, i.e. genomic tracks. By defining appropriate feature extraction approaches and similarity measures, we allow biologically meaningful clustering to be performed for genomic tracks using standard clustering algorithms. An implementation of the methodology is provided through a tool, ClusTrack, which allows fine-tuned clustering analyses to be specified through a web-based interface. We apply our methods to the clustering of occupancy of the H3K4me1 histone modification in samples from a range of different cell types. The majority of samples form meaningful subclusters, confirming that the definitions of features and similarity capture biological, rather than technical, variation between the genomic tracks. Input data and results are available, and can be reproduced, through a Galaxy Pages document at http://hyperbrowser.uio.no/hb/u/hb-superuser/p/clustrack. The clustering functionality is available as a Galaxy tool, under the menu option "Specialized analyzis of tracks", and the submenu option "Cluster tracks based on genome level similarity", at the Genomic HyperBrowser server: http://hyperbrowser.uio.no/hb/.     

Molecular Subtypes in Stage II-III Colon Cancer Defined by Genomic Instability: Early Recurrence-Risk Associated with a High Copy-Number Variation and Loss of RUNX3 and CDKN2A

Objective

We sought to investigate various molecular subtypes defined by genomic instability that may be related to early death and recurrence in colon cancer.

Methods

We sought to investigate various molecular subtypes defined by instability at microsatellites (MSI), changes in methylation patterns (CpG island methylator phenotype, CIMP) or copy number variation (CNV) in 8 genes. Stage II-III colon cancers (n = 64) were investigated by methylation-specific multiplex ligated probe amplification (MS-MLPA). Correlation of CNV, CIMP and MSI, with mutations in KRAS and BRAFV600E were assessed for overlap in molecular subtypes and early recurrence risk by uni- and multivariate regression.

Results

The CIMP phenotype occurred in 34% (22/64) and MSI in 27% (16/60) of the tumors, with noted CIMP/MSI overlap. Among the molecular subtypes, a high CNV phenotype had an associated odds ratio (OR) for recurrence of 3.2 (95% CI 1.1-9.3; P = 0.026). Losses of CACNA1G (OR of 2.9, 95% CI 1.4-6.0; P = 0.001), IGF2 (OR of 4.3, 95% CI 1.1-15.8; P = 0.007), CDKN2A (p16) (OR of 2.0, 95% CI 1.1-3.6; P = 0.024), and RUNX3 (OR of 3.4, 95% CI 1.3-8.7; P = 0.002) were associated with early recurrence, while MSI, CIMP, KRAS or BRAF V600E mutations were not. The CNV was significantly higher in deceased patients (CNV in 6 of 8) compared to survivors (CNV in 3 of 8). Only stage and loss of RUNX3 and CDKN2A were significant in the multivariable risk-model for early recurrence.

Conclusions

A high copy number variation phenotype is a strong predictor of early recurrence and death, and may indicate a dose-dependent relationship between genetic instability and outcome. Loss of tumor suppressors RUNX3 and CDKN2A were related to recurrence-risk and warrants further investigation.     

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.     

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-protein kinase A (PKA)-Csk inhibitory pathway that prevents full T-cell activation in the absence of a coreceptor stimulus. Here, we demonstrate that stimulation of CD28 leads to recruitment to lipid rafts of a β-arrestin/phosphodiesterase 4 (PDE4) complex that serves to degrade cAMP locally. Redistribution of the complex from the cytosol depends on Lck and phosphatidylinositol 3-kinase (PI3K) activity. Protein kinase B (PKB) interacts directly with β-arrestin to form part of the supramolecular complex together with sequestered PDE4. Translocation is mediated by the PKB plextrin homology (PH) domain, thus revealing a new role for PKB as an adaptor coupling PI3K and cAMP signaling. Functionally, PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production, leading to recruitment of the supramolecular PKB/β-arrestin/PDE4 complex to the membrane via the PKB PH domain, results in degradation of the TCR-induced cAMP pool located in lipid rafts, thereby allowing full T-cell activation to proceed.T-cell receptor (TCR) stimulation alone is insufficient for activation of T cells, and sustainable T-cell immune responses require a second signal in addition to the TCR-mediated signal. The second signal is typically elicited by ligands B7-1 or B7-2 on antigen-presenting cells engaging the coreceptor CD28 to prevent anergy and apoptosis and enhancing interleukin-2 (IL-2) production and clonal expansion (4). Although CD28 plays a central role in T-cell activation in vivo (5), relatively little is known about the molecular basis for the increased efficacy of T-cell activation upon TCR and CD28 costimulation. Involvement of Lck, Itk, phosphatidylinositol 3-kinase (PI3K), SLP-76, Vav-1, and phospholipase C-γ (PLC-γ) has, however, been reported (43). CD28-mediated signals are transmitted via a short intracellular stretch in the receptor containing a conserved YMNM motif (32). Phosphorylation of Tyr173 in this motif by Lck and Fyn following CD28 ligation is key to efficient signal transduction (41), generating a binding site for the SH2 domain of the p85 regulatory subunit of PI3K (37, 40). CD28 may also contribute to TCR-dependent PI3K activity without recruiting PI3K directly (18). Whether engagement of CD28 alone can also induce PI3K activity has been a matter of controversy. However, recent reports confirming phosphorylation of the protein kinase B (PKB) substrate glycogen synthase kinase 3 (GSK3) upon CD28 ligation has demonstrated that this is indeed the case (6, 15). In addition, CD28 can recruit growth factor receptor-bound protein 2 (Grb2), and such association of Grb2 occurs via the phosphorylated YMNM motif as well as via the C-terminal PXXP motif (22, 35). The PXXP motif also binds and regulates Src family kinases (SFKs) (21, 47), and knock-in mice mutated in this motif were recently reported to have impaired IL-2 secretion (16).Ligation of the TCR induces cyclic AMP (cAMP) production (27). However, the significance of this observation is still not fully understood, as it is well established that cAMP potently inhibits T-cell function and proliferation (2, 45, 46, 50). The spatiotemporal dynamics of the activation-induced cAMP gradient also are not completely appreciated. We have previously shown that cAMP is rapidly produced in lipid rafts following engagement of the TCR in primary T cells (3). This activates a pool of PKA type I targeted to rafts by association with the anchoring protein Ezrin, forming part of a supramolecular complex where Ezrin, EBP50, and PAG provide a scaffold that is able to coordinate PKA phosphorylation and activation of Csk, thereby inhibiting T-cell activation (44, 50). In addition, we have demonstrated that CD3/CD28 costimulation leads to recruitment of type 4 phosphodiesterase (PDE4) isoforms to rafts, resulting in degradation of the TCR-induced cAMP pool (3). Thus, we envisage that TCR-induced cAMP production constitutes a negative feedback loop capable of abrogating T-cell activation in the absence of a second signal. In order then to allow full T-cell activation to proceed, cAMP-mediated inhibition must be lifted. This appears to occur in the presence of a costimulus involving CD28 acting to trigger recruitment of PDE4 to lipid rafts, thereby degrading cAMP at this spatially critical location and resulting in an overriding positive feed-forward signal rather than the negative feedback loop activated from the TCR. In addition, a recent publication by Conche et al. has also found a possible stimulatory effect of cAMP, as the paper surprisingly showed that a transient cAMP increase shortly after TCR triggering may potentiate the calcium component of the TCR signaling. This could constitute a positive feed-forward in addition to the negative feedback signal by cAMP (12).Spatial organization and recruitment of mediators of specific pathways as outlined above are essential to ensure signaling specificity and amplification. Among the many protein scaffolds linking effector molecules into linear pathways, β-arrestins have been reported to confer cross talk with a growing list of molecules important in cellular trafficking and signal transduction, including Src family members and mitogen-activated protein (MAP) kinases (reviewed in reference 14). The arrestins were first identified as having a role in desensitization of G protein-coupled receptors (GPCRs) (9); later, they were discovered to be involved in receptor internalization by interacting with clathrin and AP-2, thereby bringing activated receptors to clathrin-coated pits for endocytosis (19, 26). A role for β-arrestin in the spatially localized degradation of cAMP by scaffolding PDE4 isoforms to the proximity of cAMP generation at the plasma membrane has also been suggested (3, 7, 30, 38).In the present study, we uncover a novel pathway that defines how T-cell costimulation elicits recruitment of PDE4 to lipid rafts to overcome cAMP-mediated inhibition of T-cell activation. This pathway is initiated by CD28 engagement leading to PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production and resulting in recruitment of a supramolecular complex of PKB/β-arrestin/PDE4 targeted to the plasma membrane due to sequestration via the PKB plextrin homology (PH) domain. Functionally, this pathway is essential for CD28 costimulation to strengthen and sustain T-cell immune responses.     

Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

Background  

The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH₄ prolactin producing cell line from rat, and primary cell culture from fish pituitaries.     

Intestinal barrier function of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator

Background  

Fish farmed under high intensity aquaculture conditions are subjected to unnatural environments that may cause stress. Therefore awareness of how to maintain good health and welfare of farmed fish is important. For Atlantic salmon held in sea cages, water flow, dissolved oxygen (DO) levels and temperature will fluctuate over time and the fish can at times be exposed to detrimentally low DO levels and high temperatures. This experimental study investigates primary and secondary stress responses of Atlantic salmon post smolts to long-term exposure to reduced and fluctuating DO levels and high water temperatures, mimicking situations in the sea cages. Plasma cortisol levels and cortisol release to the water were assessed as indicators of the primary stress response and intestinal barrier integrity and physiological functions as indicators of secondary responses to changes in environmental conditions.     

DNA-uracil and human pathology

Uracil is usually an inappropriate base in DNA, but it is also a normal intermediate during somatic hypermutation (SHM) and class switch recombination (CSR) in adaptive immunity. In addition, uracil is introduced into retroviral DNA by the host as part of a defence mechanism. The sources of uracil in DNA are spontaneous or enzymatic deamination of cytosine (U:G mispairs) and incorporation of dUTP (U:A pairs). Uracil in DNA is removed by a uracil-DNA glycosylase. The major ones are nuclear UNG2 and mitochondrial UNG1 encoded by the UNG-gene, and SMUG1 that also removes oxidized pyrimidines, e.g. 5-hydroxymethyluracil. The other ones are TDG that removes U and T from mismatches, and MBD4 that removes U from CpG contexts. UNG2 is found in replication foci during the S-phase and has a distinct role in repair of U:A pairs, but it is also important in U:G repair, a function shared with SMUG1. SHM is initiated by activation-induced cytosine deaminase (AID), followed by removal of U by UNG2. Humans lacking UNG2 suffer from recurrent infections and lymphoid hyperplasia, and have skewed SHM and defective CSR, resulting in elevated IgM and strongly reduced IgG, IgA and IgE. UNG-defective mice also develop B-cell lymphoma late in life. In the defence against retrovirus, e.g. HIV-1, high concentrations of dUTP in the target cells promotes misincorporation of dUMP-, and host cell APOBEC proteins may promote deamination of cytosine in the viral DNA. This facilitates degradation of viral DNA by UNG2 and AP-endonuclease. However, viral proteins Vif and Vpr counteract this defense by mechanisms that are now being revealed. In conclusion, uracil in DNA is both a mutagenic burden and a tool to modify DNA for diversity or degradation.     

Gene, phenotype and function: GLABROUS1 and resistance to herbivory in natural populations of Arabidopsis lyrata

The molecular genetic basis of adaptive variation is of fundamental importance for evolutionary dynamics, but is still poorly known. Only in very few cases has the relationship between genetic variation at the molecular level, phenotype and function been established in natural populations. We examined the functional significance and genetic basis of a polymorphism in production of leaf hairs, trichomes, in the perennial herb Arabidopsis lyrata. Earlier studies suggested that trichome production is subject to divergent selection. Here we show that the production of trichomes is correlated with reduced damage from insect herbivores in natural populations, and using statistical methods developed for medical genetics we document an association between loss of trichome production and mutations in the regulatory gene GLABROUS1. Sequence data suggest that independent mutations in this regulatory gene have provided the basis for parallel evolution of reduced resistance to insect herbivores in different populations of A. lyrata and in the closely related Arabidopsis thaliana. The results show that candidate genes identified in model organisms provide a valuable starting point for analysis of the genetic basis of phenotypic variation in natural populations.     

Food web dynamics affect Northeast Arctic cod recruitment

Proper management of ecosystems requires an understanding of both the species interactions as well as the effect of climate variation. However, a common problem is that the available time-series are of different lengths. Here, we present a general approach for studying the dynamic structure of such interactions. Specifically, we analyse the recruitment of the world's largest cod stock, the Northeast Arctic cod. Studies based on data starting in the 1970-1980s indicate that this stock is affected by temperature through a variety of pathways. However, the value of such studies is somewhat limited by the fact that they are based on a quite specific ecological and climatic situation. Recently, this stock has consisted of fairly young fish and the spawning stock has consisted of relatively few age groups. In this study, we develop a model for the effect of capelin (the cod's main prey) and herring on cod recruitment since 1973. Based on this model, we analyse data on cod, herring and temperature going back to 1921 and find that food-web effects explain a significant part of the cod recruitment variation back to around 1950.