Update of the FANTOM web resource: from mammalian transcriptional landscape to its dynamic regulation.

The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. The primary and unique technique is cap analysis of gene expression (CAGE), sequencing mRNA 5'-ends with a second-generation sequencer to quantify promoter activities even in the absence of gene annotation. Additional genome-wide experiments complement the setup including short RNA sequencing, microarray gene expression profiling on large-scale perturbation experiments and ChIP-chip for epigenetic marks and transcription factors. All the experiments are performed in a differentiation time course of the THP-1 human leukemic cell line. Furthermore, we performed a large-scale mammalian two-hybrid (M2H) assay between transcription factors and monitored their expression profile across human and mouse tissues with qRT-PCR to address combinatorial effects of regulation by transcription factors. These interdependent data have been analyzed individually and in combination with each other and are published in related but distinct papers. We provide all data together with systematic annotation in an integrated view as resource for the scientific community (http://fantom.gsc.riken.jp/4/). Additionally, we assembled a rich set of derived analysis results including published predicted and validated regulatory interactions. Here we introduce the resource and its update after the initial release.

Are there positive emotions in short-term dynamic psychotherapy or is it all Freude-less ?

Psychodynamic therapists are often suspicious of positive emotions and consider them to be nothing more than a form of denial or of another defense aiming to diminish painful or difficult affects. Positive emotions seem to exist only through the absence of negative emotions or as something that may happen outside of therapy. On the other hand, clinicians also agree that psychoanalytic work could not be successful without such positive emotions as interest, pleasure, surprise and creativity. Contemporary psychoanalytic thinking and new research findings in the area of relationship regulation are likely to give positive emotions an increasingly prominent place in dynamically oriented therapies. With today's emphasis on the therapeutic relationship and intersubjectivity, the time appears right to integrate positive emotions more formally into psychodynamic clinical theories.

Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARalpha helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.

A dynamic view of hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle.

Status epilepticus: an independent outcome predictor after cerebral anoxia.

BACKGROUND: Prognosis of status epilepticus (SE) depends on its cause, but there is uncertainty as to whether SE represents an independent outcome predictor for a given etiology. Cerebral anoxia is a relatively homogenous severe encephalopathy. Postanoxic SE is associated to a nearly 100% mortality in this setting; however, it is still unclear whether this is a severity marker of the underlying encephalopathy, or an independent factor influencing outcome. The goal of this study was to assess if postanoxic SE is independently associated with mortality after cerebral anoxia. METHODS: This was a retrospective observation of consecutive comatose survivors of cardiac arrest, including subjects treated with hypothermia. On the subgroup with EEG recordings in the first hospitalization days, univariate and multivariate analyses were applied to potential determinants of in-hospital mortality, and included the following variables: age, gender, type and length of cardiac arrest, occurrence of circulatory shock, presence of therapeutic hypothermia, and electrographic SE. RESULTS: Out of 166 postanoxic patients, 107 (64%) had an EEG (median latency from admission, 2 days); in this group, therapeutic hypothermia was administered in 59%. Death occurred in 71 (67%) patients. Postanoxic SE was associated with mortality regardless of type of acute cardiac rhythm and administration of hypothermic treatment. CONCLUSION: In this hospital-based cohort, postanoxic status epilepticus (SE) seems to be independently related to death in cardiac arrest survivors, suggesting that SE might determine a bad prognosis for a given etiology. Confirmation of these results in a prospective assessment is needed.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

A combined molecular clinical predictor of survival validated with the rtog-0525 cohort

For glioblastoma (GBM), survival classification has primarily relied on clinical criteria, exemplified by the Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA). We sought to improve tumor classification by combining tumor biomarkers with the clinical RPA data. To accomplish this, we first developed 4 molecular biomarkers derived from gene expression profiling, a glioma CpG island methylator phenotype, a novel MGMT promoter methylation assay, and IDH1 mutations. A molecular predictor (MP) model was created with these 4 biomarkers on a training set of 220 retrospectively collected archival GBMtumors. ThisMPwas further combined with RPA classification to develop a molecular-clinical predictor (MCP). The median survivals for the combined, 4-class MCP were 65 months, 31 months, 13 months, and 9 months, which was significantly improved when compared with the RPA alone. The MCP was then applied to 725 samples from the RTOG-0525 cohort, showing median survival for each risk group of NR, 26 months, 16 months, and 11 months. The MCP was significantly improved over the RPA at outcome prediction in the RTOG 0525 cohort with a 33%increase in explained variation with respect to survival, validating the result obtained in the training set. To illustrate the benefit of the MCP for patient stratification, we examined progression-free survival (PFS) for patients receiving standard-dose temozolomide (SD-TMZ) vs. dose-dense TMZ (DD-TMZ) in RPA and MCP risk groups. A significant difference between DD-TMZ and SD-TMZ was observed in the poorest surviving MCP risk group with a median PFS of 6 months vs. 3 months (p ¼ 0.048, log-rank test). This difference was not seen using the RPA classification alone. In summary, we have developed a combined molecular-clinical predictor that appears to improve outcome prediction when compared with clinical variables alone. This MCP may serve to better identify patients requiring intensive treatments beyond the standard of care.

The role of energetic value in dynamic brain response adaptation during repeated food image viewing.

The repeated presentation of simple objects as well as biologically salient objects can cause the adaptation of behavioral and neural responses during the visual categorization of these objects. Mechanisms of response adaptation during repeated food viewing are of particular interest for better understanding food intake beyond energetic needs. Here, we measured visual evoked potentials (VEPs) and conducted neural source estimations to initial and repeated presentations of high-energy and low-energy foods as well as non-food images. The results of our study show that the behavioral and neural responses to food and food-related objects are not uniformly affected by repetition. While the repetition of images displaying low-energy foods and non-food modulated VEPs as well as their underlying neural sources and increased behavioral categorization accuracy, the responses to high-energy images remained largely invariant between initial and repeated encounters. Brain mechanisms when viewing images of high-energy foods thus appear less susceptible to repetition effects than responses to low-energy and non-food images. This finding is likely related to the superior reward value of high-energy foods and might be one reason why in particular high-energetic foods are indulged although potentially leading to detrimental health consequences.

Change in defense mechanisms and coping over the course of short-term dynamic psychotherapy for adjustment disorder.

Short-term dynamic psychotherapy (STDP) has rarely been investigated with regard to its underlying mechanisms of change, even if psychoanalytic theory informs us about several potential putative mechanisms of change in patients. Change in overall defensive functioning is one. In this study, we explored the role of overall defensive functioning, by comparing it on the process level with the neighbouring concept of overall coping functioning. A total of N=32 patients, mainly presenting adjustment disorder, were included in the study. The patients underwent STDP up to 40 sessions; three sessions per psychotherapy were transcribed and analyzed by using two observer-rating scales: Defense Mechanism Rating Scales (Perry, 1990) and Coping Action Patterns (Perry, Drapeau, Dunkley, & Blake, 2005). Hierarchical linear modeling was applied to model the change over the course of therapy and relate it to outcome. Results suggest that STDP has an effect on the target variable of overall defensive functioning, which was absent for overall coping functioning. Links with outcome confirm the importance of the effect. These results are discussed from methodological and clinical viewpoints.

Interferon-Induced Gene Expression Is a Stronger Predictor of Treatment Response Than IL28B Genotype in Patients With Hepatitis C.

BACKGROUND & AIMS: The host immune response during the chronic phase of hepatitis C virus infection varies among individuals; some patients have a no interferon (IFN) response in the liver, whereas others have full activation IFN-stimulated genes (ISGs). Preactivation of this endogenous IFN system is associated with nonresponse to pegylated IFN-α (pegIFN-α) and ribavirin. Genome-wide association studies have associated allelic variants near the IL28B (IFNλ3) gene with treatment response. We investigated whether IL28B genotype determines the constitutive expression of ISGs in the liver and compared the abilities of ISG levels and IL28B genotype to predict treatment outcome. METHODS: We genotyped 109 patients with chronic hepatitis C for IL28B allelic variants and quantified the hepatic expression of ISGs and of IL28B. Decision tree ensembles, in the form of a random forest classifier, were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele was significantly associated with increased expression of ISG. However, stratification of the patients according to treatment response revealed increased ISG expression in nonresponders, irrespective of IL28B genotype. Multivariate analysis of ISG expression, IL28B genotype, and several other factors associated with response to therapy identified ISG expression as the best predictor of treatment response. CONCLUSIONS: IL28B genotype and hepatic expression of ISGs are independent predictors of response to treatment with pegIFN-α and ribavirin in patients with chronic hepatitis C. The most accurate prediction of response was obtained with a 4-gene classifier comprising IFI27, ISG15, RSAD2, and HTATIP2.

Principal components of functional connectivity: A new approach to study dynamic brain connectivity during rest.

Functional connectivity (FC) as measured by correlation between fMRI BOLD time courses of distinct brain regions has revealed meaningful organization of spontaneous fluctuations in the resting brain. However, an increasing amount of evidence points to non-stationarity of FC; i.e., FC dynamically changes over time reflecting additional and rich information about brain organization, but representing new challenges for analysis and interpretation. Here, we propose a data-driven approach based on principal component analysis (PCA) to reveal hidden patterns of coherent FC dynamics across multiple subjects. We demonstrate the feasibility and relevance of this new approach by examining the differences in dynamic FC between 13 healthy control subjects and 15 minimally disabled relapse-remitting multiple sclerosis patients. We estimated whole-brain dynamic FC of regionally-averaged BOLD activity using sliding time windows. We then used PCA to identify FC patterns, termed "eigenconnectivities", that reflect meaningful patterns in FC fluctuations. We then assessed the contributions of these patterns to the dynamic FC at any given time point and identified a network of connections centered on the default-mode network with altered contribution in patients. Our results complement traditional stationary analyses, and reveal novel insights into brain connectivity dynamics and their modulation in a neurodegenerative disease.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.