High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Leadership at the upper echelons : three essays

"IT'S THE ECONOMY STUPID", BUT CHARISMA MATTERS TOO: A DUAL PROCESS MODEL OF PRESIDENTIAL ELECTION OUTCOMES. ABSTRACT Because charisma is assumed to be an important determinant of effective leadership, the extent to which a presidential nominee is more charismatic than his opponent should be an important determinant of voter choices. We computed a composite measure of the rhetorical richness of acceptances speeches given by U.S. presidential candidates at their national party convention. We added this marker of charisma to Ray C. Fair's presidential vote-share equation (1978; 2009). We theorized that voters decide using psychological attribution (i.e., due to macroeconomics and incumbency) as well as inferential processes (i.e., due to leader charismatic behavior) when voting. Controlling for the macro-level variables and incumbency in the Fair model, our results indicated that difference between nominees' charisma is a significant determinant of electoral success, particularly in close elections. This extended model significantly improves the precision of the Fair model and correctly predicts 23 out of the last 24 U.S. presidential elections. Paper 2: IT CEO LEADERSHIP, CORPORATE SOCIAL AND FINANCIAL PERFORMANCE. ABSTRACT We investigated whether CEO leadership predicted corporate financial performance (CFP) and corporate social performance (CSP). Using longitudinal data on 258 CEOs from 117 firms across 19 countries and 10 industry sectors, we found that determinants of CEO leadership (i.e., implicit motives) significantly predicted both CFP and CSP. As expected, the most consistent positive predictor was Responsibility Disposition when interacting with n (need for) Power. n Achievement and n Affiliation were generally negatively related or unrelated to outcomes. CSP was positively related to accounting measures of CFP. Our findings suggest that executive leader characteristics have important consequences for corporate level outcomes. Paper 3. PUNISHING THE POWERFUL: ATTRIBUTIONS OF BLAME AND LEADERSHIP ABSTRACT We propose that individuals are more lenient in attributing blame to leaders than to nonleaders. We advance a motivational explanation building on the perspective of punishment and on system justification theory. We conducted two scenario experiments which supported our proposition. In study 1, wrongdoer leader status was negatively related to blame and the perceived seriousness of the wrongdoing. In study 2, controlling for the Big-Five personality factor and individual differences in moral evaluation (i.e., moral foundations), wrongdoer leader status was negatively related with desired severity of punishment, and fair punishments were perceived as more just for non-leaders than for leaders.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

The αVβ3/αVβ5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model.

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Comparative localization of glioma-reactive monoclonal antibodies in vivo in an athymic mouse human glioma xenograft model.

Radioiodinated murine monoclonal antibodies (Mabs) 81C6, Me 1-14, C12, D12, and E9, made against or reactive with human gliomas but not normal brain, and Mab UJ13A, a pan-neuroectodermal Mab reactive with normal human glial and neural cells, were evaluated in paired label studies in the D-54 MG subcutaneous human glioma xenograft model system in nude mice. Following intravenous injection in the tail vein of mice bearing 200-400 mm3 tumors, specific localization of Mabs to tumor over time (6 h-9 days) was evaluated by tissue counting; each Mab demonstrated a unique localization profile. The comparison of localization indices (LI), determined as a ratio of tissue level of Mab to control immunoglobulin with simultaneous correction for blood levels of each, showed Mabs 81C6 and Me 1-14 to steadily accumulate in glioma xenografts, maintaining LI from 5-20 at 7-9 days after Mab injection. Mab UJ13A peaked at day 1, maintaining this level through day 2, and declining thereafter. Mabs D12 and C12 peaked at days 3 and 4, respectively, and E9 maintained an LI of greater than 3 from days 3-9. Percent injected dose localized/g of tumor varied from a peak high of 16% (81C6) to a low of 5% (Me 1-14 and UJ13A). Immunoperoxidase histochemistry, performed with each Mab on a battery of primary human brain neoplasms, revealed that Mabs 81C6 and E9, which demonstrated the highest levels of percent injected dose localized/g of tumor over time, reacted with antigens expressed in the extracellular matrix. This finding suggests that extracellular matrix localization of antigen represents a biologically significant factor affecting localization and/or binding in the xenograft model used. The demonstration of significant localization, varied kinetics and patterns of localization of this localizing Mab panel warrants their continued investigation as potential imaging and therapeutic agents for human trials.

The ability model of emotional intelligence: Searching for valid measures

Current measures of ability emotional intelligence (EI)--including the well-known Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT)--suffer from several limitations, including low discriminant validity and questionable construct and incremental validity. We show that the MSCEIT is largely predicted by personality dimensions, general intelligence, and demographics having multiple R's with the MSCEIT branches up to .66; for the general EI factor this relation was even stronger (Multiple R = .76). As concerns the factor structure of the MSCEIT, we found support for four first-order factors, which had differential relations with personality, but no support for a higher-order global EI factor. We discuss implications for employing the MSCEIT, including (a) using the single branches scores rather than the total score, (b) always controlling for personality and general intelligence to ensure unbiased parameter estimates in the EI factors, and (c) correcting for measurement error. Failure to account for these methodological aspects may severely compromise predictive validity testing. We also discuss avenues for the improvement of ability-based tests.

A comparison of three methods of Mendelian randomization when the genetic instrument, the risk factor and the outcome are all binary.

The method of instrumental variable (referred to as Mendelian randomization when the instrument is a genetic variant) has been initially developed to infer on a causal effect of a risk factor on some outcome of interest in a linear model. Adapting this method to nonlinear models, however, is known to be problematic. In this paper, we consider the simple case when the genetic instrument, the risk factor, and the outcome are all binary. We compare via simulations the usual two-stages estimate of a causal odds-ratio and its adjusted version with a recently proposed estimate in the context of a clinical trial with noncompliance. In contrast to the former two, we confirm that the latter is (under some conditions) a valid estimate of a causal odds-ratio defined in the subpopulation of compliers, and we propose its use in the context of Mendelian randomization. By analogy with a clinical trial with noncompliance, compliers are those individuals for whom the presence/absence of the risk factor X is determined by the presence/absence of the genetic variant Z (i.e., for whom we would observe X = Z whatever the alleles randomly received at conception). We also recall and illustrate the huge variability of instrumental variable estimates when the instrument is weak (i.e., with a low percentage of compliers, as is typically the case with genetic instruments for which this proportion is frequently smaller than 10%) where the inter-quartile range of our simulated estimates was up to 18 times higher compared to a conventional (e.g., intention-to-treat) approach. We thus conclude that the need to find stronger instruments is probably as important as the need to develop a methodology allowing to consistently estimate a causal odds-ratio.

Temperature-dependent turnovers in sex-determination mechanisms: a quantitative model.

Sex determination is often seen as a dichotomous process: individual sex is assumed to be determined either by genetic (genotypic sex determination, GSD) or by environmental factors (environmental sex determination, ESD), most often temperature (temperature sex determination, TSD). We endorse an alternative view, which sees GSD and TSD as the ends of a continuum. Both effects interact a priori, because temperature can affect gene expression at any step along the sex-determination cascade. We propose to define sex-determination systems at the population- (rather than individual) level, via the proportion of variance in phenotypic sex stemming from genetic versus environmental factors, and we formalize this concept in a quantitative-genetics framework. Sex is seen as a threshold trait underlain by a liability factor, and reaction norms allow modeling interactions between genotypic and temperature effects (seen as the necessary consequences of thermodynamic constraints on the underlying physiological processes). As this formalization shows, temperature changes (due to e.g., climatic changes or range expansions) are expected to provoke turnovers in sex-determination mechanisms, by inducing large-scale sex reversal and thereby sex-ratio selection for alternative sex-determining genes. The frequency of turnovers and prevalence of homomorphic sex chromosomes in cold-blooded vertebrates might thus directly relate to the temperature dependence in sex-determination mechanisms.

Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0.

In the plant-beneficial, root-colonizing strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively regulates the synthesis of biocontrol factors (mostly antifungal secondary metabolites) and contributes to oxidative stress response via the stress sigma factor RpoS. The backbone of this pathway consists of the GacS/GacA two-component system, which activates the expression of three small regulatory RNAs (RsmX, RsmY, RsmZ) and thereby counters translational repression exerted by the RsmA and RsmE proteins on target mRNAs encoding biocontrol factors. We found that the expression of typical biocontrol factors, that is, antibiotic compounds and hydrogen cyanide (involving the phlA and hcnA genes), was significantly lower at 35 degrees C than at 30 degrees C. The expression of the rpoS gene was affected in parallel. This temperature control depended on RetS, a sensor kinase acting as an antagonist of the GacS/GacA system. An additional sensor kinase, LadS, which activated the GacS/GacA system, apparently did not contribute to thermosensitivity. Mutations in gacS or gacA were epistatic to (that is, they overruled) mutations in retS or ladS for expression of the small RNAs RsmXYZ. These data are consistent with a model according to which RetS-GacS and LadS-GacS interactions shape the output of the Gac/Rsm pathway and the environmental temperature influences the RetS-GacS interaction in P. fluorescens CHA0.

The four- vs. alternative six-factor structure of the French WISC-IV: Comparison using confirmatory factor analyses

Exploratory and confirmatory factor analyses reported in the French technical manual of the WISC-IV provides evidence supporting a structure with four indices: Verbal Comprehension (VCI), Perceptual Reasoning (PRI), Working Memory (WMI), and Processing Speed (PSI). Although the WISC-IV is more attuned to contemporary theory, it is still not in total accordance with the dominant theory: the Cattell-Horn-Carroll (CHC) theory of cognitive ability. This study was designed to determine whether the French WISC-IV is better described with the four-factor solution or whether an alternative model based on the CHC theory is more appropriate. The intercorrelations matrix reported in the French technical manual was submitted to confirmatory factor analysis. A comparison of competing models suggests that a model based on the CHC theory fits the data better than the current WISC-IV structure. It appears that the French WISC-IV in fact measures six factors: crystallized intelligence (Gc), fluid intelligence (Gf), short-term memory (Gsm), processing speed (Gs), quantitative knowledge (Gq), and visual processing (Gv). We recommend that clinicians interpret the subtests of the French WISC-IV in relation to this CHC model in addition to the four indices.

Decreased behavioral impairments in an Alzheimer mice model by interfering with TNF-alpha metabolism.

The performance of mice expressing PDAPP (+/+ or +/-) was studied in the Morris place navigation task. Different lines of questions were investigated using PDAPP+/- mice in which the activity of the cytokine Tumor Necrosing Factor alpha (TNFalpha) was attenuated by chronic treatment with anti-TNF or deleting TNFalpha (TNF-/-). Two different categories of behavior were analyzed in adult (6 months) and middle aged (15 months) subjects. Classically, the cognitive performance was assessed from the escape efficacy and quantitative bias toward the training position in a Morris water maze. Second, stereotyped circling was quantified, along with more qualitative behavioral impairments such as self-mutilation or increased reactivity. Our results can be summarized as follows. (1) All of the PDAPP mice expressed reduced cognitive performance in the Morris task, but only those with a clear-cut amyloid burden in the hippocampus showed behavioral abnormalities such as stereotyped circling. (2) Chronic treatment with anti-TNF prevented the development of pathological circling in the 6-month-old mice but not in the 15-month-old mice and had no significant effect on amyloid burden. (3) The absence of TNFalpha prevented the development of stereotyped circling in 6- and 15-month-old mice but increased amyloid burden after 15 months. These data indicate that PDAPP mice express cognitive impairments disregarding absence of TNF. The pathological behavioral anomalies related to the PDAPP mutation seem reduced by treatments interfering with TNFalpha.

The δ-Opioid Receptor Affects Epidermal Homeostasis via ERK-Dependent Inhibition of Transcription Factor POU2F3.

Neuropeptides and their receptors are present in human skin, and their importance for cutaneous homeostasis and during wound healing is increasingly appreciated. However, there is currently a lack of understanding of the molecular mechanisms by which their signaling modulates keratinocyte function. Here, we show that δ-opioid receptor (DOPr) activation inhibits proliferation of human keratinocytes, resulting in decreased epidermal thickness in an organotypic skin model. DOPr signaling markedly delayed induction of keratin intermediate filament (KRT10) during in vitro differentiation and abolished its induction in the organotypic skin model. This was accompanied by deregulation of involucrin (IVL), loricrin, and filaggrin. Analysis of the transcription factor POU2F3, which is involved in regulation of KRT10, IVL, and profilaggrin expression, revealed a DOPr-mediated extracellular signal-regulated kinase (ERK)-dependent downregulation of this factor. We propose that DOPr signaling specifically activates the ERK 1/2 mitogen-activated protein kinase pathway to regulate keratinocyte functions. Complementing our earlier studies in DOPr-deficient mice, these data suggest that DOPr activation in human keratinocytes profoundly influences epidermal morphogenesis and homeostasis.

Distribution of free and liposomal doxorubicin after isolated lung perfusion in a sarcoma model.

BACKGROUND: Isolated lung perfusion (ILP) with free and a novel liposomal-encapsulated doxorubicin (Liporubicin, CT Sciences SA, Lausanne, Switzerland) was compared with respect to drug uptake and distribution in rat lungs bearing a sarcomatous tumor. METHODS: A single sarcomatous tumor was generated in the left lung of 39 Fischer rats, followed 10 days later by left-sided ILP (n = 36) with free and equimolar-dosed liposomal doxorubicin at doses of 100 microg (n = 9) and 400 microg (n = 9) for each doxorubicin formulation. In each perfused lung, the drug concentration and distribution were assessed in the tumor and in three areas of normal lung parenchyma by high-performance liquid chromatography (n = 6) and fluorescence microscopy (n = 3). Histologic assessment and immunostaining with von Willebrand factor was performed in 3 animals with untreated tumors. RESULTS: The sarcomatous tumors in controls were well vascularized with fine branching capillaries present throughout the tumors. Isolated lung perfusion resulted in a heterogeneous drug distribution within the perfused lung and a consistently lower drug uptake in tumors than in lung parenchyma for both doxorubicin formulations and both drug doses applied. Isolated lung perfusion with free doxorubicin resulted in a significantly higher drug uptake than Liporubicin in both the tumor and lung tissue for both drug doses applied (p < 0.01). However, the tumor/normal tissue drug ratio was lower for free than for liposomal doxorubicin at a drug dose of 100 microg (0.27 +/- 0.1 vs 0.53 +/- 0.5; p = 0.225) and similar for both doxorubicin formulations at a drug dose of 400 microg (0.67 +/- 0.2 vs 0.54 +/- 0.2; p = 0.335). Both doxorubicin formulations resulted in fluorescence signaling emerging from all tissue compartments of normal lung parenchyma but only in weak and sporadic signaling from the tumors confined to the tumor periphery and vessels situated within the tumor for both drug doses assessed. CONCLUSIONS: Isolated lung perfusion with free and liposomal doxorubicin resulted in a heterogeneous drug distribution within the perfused lung and in a lower drug uptake in tumors than in lung tissue for both doxorubicin formulations and drug doses applied.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.