Phosphorylation sites of protein kinase C δ in H2O2-treated cells and its activation by tyrosine kinase in vitro

Protein kinase C δ (PKC δ) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC δ in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC δ in response to H2O2.

RNA virus error catastrophe: Direct molecular test by using ribavirin

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.

To test whether the structure of a protein is determined in a manner akin to the assembly of a jigsaw puzzle, up to 10 adjacent residues within the core of T4 lysozyme were replaced by methionine. Such variants are active and fold cooperatively with progressively reduced stability. The structure of a seven-methionine variant has been shown, crystallographically, to be similar to wild type and to maintain a well ordered core. The interaction between the core residues is, therefore, not strictly comparable with the precise spatial complementarity of the pieces of a jigsaw puzzle. Rather, a certain amount of give and take in forming the core structure is permitted. A simplified hydrophobic core sequence, imposed without genetic selection or computer-based design, is sufficient to retain native properties in a globular protein.

Molecular phylogeny analysis of fiddler crabs: test of the hypothesis of increasing behavioral complexity in evolution.

The current phylogenetic hypothesis for the evolution and biogeography of fiddler crabs relies on the assumption that complex behavioral traits are assumed to also be evolutionary derived. Indo-west Pacific fiddler crabs have simpler reproductive social behavior and are more marine and were thought to be ancestral to the more behaviorally complex and more terrestrial American species. It was also hypothesized that the evolution of more complex social and reproductive behavior was associated with the colonization of the higher intertidal zones. Our phylogenetic analysis, based upon a set of independent molecular characters, however, demonstrates how widely entrenched ideas about evolution and biogeography led to a reasonable, but apparently incorrect, conclusion about the evolutionary trends within this pantropical group of crustaceans. Species bearing the set of "derived traits" are phylogenetically ancestral, suggesting an alternative evolutionary scenario: the evolution of reproductive behavioral complexity in fiddler crabs may have arisen multiple times during their evolution. The evolution of behavioral complexity may have arisen by coopting of a series of other adaptations for high intertidal living and antipredator escape. A calibration of rates of molecular evolution from populations on either side of the Isthmus of Panama suggest a sequence divergence rate for 16S rRNA of 0.9% per million years. The divergence between the ancestral clade and derived forms is estimated to be approximately 22 million years ago, whereas the divergence between the American and Indo-west Pacific is estimated to be approximately 17 million years ago.

The [(G/C)3NN]n motif: a common DNA repeat that excludes nucleosomes.

Nucleosomes, the basic structural elements of chromosomes, consist of 146 bp of DNA coiled around an octamer of histone proteins, and their presence can strongly influence gene expression. Considerations of the anisotropic flexibility of nucleotide triplets containing 3 cytosines or guanines suggested that a [5'(G/C)3 NN3']n motif might resist wrapping around a histone octamer. To test this, DNAs were constructed containing a 5'-CCGNN-3' pentanucleotide repeat with the Ns varied. Using in vitro nucleosome reconstitution and electron microscopy, a plasmid with 48 contiguous CCGNN repeats strongly excluded nucleosomes in the repeat region. Competitive reconstitution gel retardation experiments using DNA fragments containing 12, 24, or 48 CCGNN repeats showed that the propensity to exclude nucleosomes increased with the length of the repeat. Analysis showed that a 268-bp DNA containing a (CCGNN)48 block is 4.9 +/- 0.6-fold less efficient in nucleosome assembly than a similar length pUC19 fragment and approximately 78-fold less efficient than a similar length (CTG)n sequence, based on results from previous studies. Computer searches against the GenBank database for matches with a [(G/C)3NN]48 sequence revealed numerous examples that frequently were present in the control regions of "TATA-less" genes, including the human ETS-2 and human dihydrofolate reductase genes. In both cases the (G/C)3NN repeat, present in the promoter region, co-maps with loci previously shown to be nuclease hypersensitive sites.

Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography.

Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learning-related activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in brain-damaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.

La atención sostenida como marcador de vulnerabilidad de la esquizofrenia: una serie de Meta-análisis de estudios con Test de Ejecución Continua (CPT

Los déficit cognitivos son una característica nuclear de la esquizofrenia (Heinrichs, 2005). En la actualidad, su estudio ha cobrado una gran relevancia debido a su estrecha relación con el funcionamiento de las personas con esquizofrenia (Malhotra y Schooler, 2014) y a su uso como marcadores de vulnerabilidad y endofenotipos de este trastorno (Ritsner y Gottesman, 2011). La atención sostenida, evaluada con Tareas de Ejecución Continua (CPT; Rosvold et al., 1956), es una de las principales áreas afectadas en la esquizofrenia (Cornblatt y Keilp, 1994; Nuechterlein, 1991). Numerosos estudios han mostrado que las personas con esquizofrenia tienen un rendimiento deficitario en estas tareas en comparación con controles normales, que los déficit en la atención sostenida son estables e independientes de factores clínicos, y que están presentes en sus familiares. En el marco de los modelos de vulnerabilidad-estrés (Zubin y Spring, 1977; Nuechterlein et al., 1992), los déficit en la atención sostenida medidos con CPT han sido propuestos como marcador de vulnerabilidad y endofenotipo del trastorno (Cornblatt y Malhotra, 2001; Gur et al., 2007; Greenwood et al., 2013). Hasta la fecha, ningún estudio ha integrado cuantitativamente los resultados de estudios sobre la atención sostenida evaluada con CPT en la esquizofrenia. El objetivo principal de este trabajo fue integrar la evidencia empírica existente sobre déficit en la atención sostenida medida con tareas CPT en la esquizofrenia mediante técnicas de meta-análisis. La hipótesis central planteada es que estos déficit son marcadores de vulnerabilidad, al reunir los siguientes criterios: a) Están presentes en personas con esquizofrenia en mayor magnitud que en controles normales; b) Son independientes de variables clínicas; c) Son específicos; su magnitud es mayor en comparación con otros grupos clínicos; d) Están presentes en sus familiares en mayor magnitud que en controles normales...

Recuperación de estructuras latentes y estimación de parámetros en test multidimensionales ordinales: análisis comparado entre la TRI la TCT y el análisis factorial

A través de la realización de cuatro estudios basados en datos simulados y unidos por una preocupación común, aunque parcialmente independientes en su metodología y foco específico, esta tesis se plantea como objetivo general poner a prueba algunas de las ventajas que podría obtener un investigador aplicado al utilizar modelos clásicos de TRI frente a otras opciones, para de esta forma evaluar la posibilidad de obtener resultados relativamente equivalentes utilizando procedimientos alternativos. Específicamente, en esta tesis se pone a prueba: a) la calidad diferencial de las estimaciones de las propiedades de los ítems obtenidas por los procedimientos TRI frente a los factoriales; b) la validez de los procedimientos de escalamiento TRI frente a los de la TCT; y c) los límites de la propiedad de invarianza de las estimaciones de sujetos e ítems obtenidas con procedimientos TRI. La hipótesis general de esta tesis es que, en el tipo de condiciones en que se ha enfocado el estudio, las ventajas de utilizar modelos clásicos de TRI no serán demasiado amplias, con lo que existirán condiciones en las que emplear procedimientos alternativos será recomendable, dada su simplicidad relativa y su equivalencia de resultados. La primera investigación utiliza datos simulados para comparar las estimaciones de discriminación de ítems politómicos provenientes del AFI, frente a las posibles de obtener con distintas estrategias relacionadas con el análisis factorial clásico. La segunda investigación, también utiliza datos simulados para poner a prueba la equivalencia de las estimaciones de discriminación de los ítems obtenidas con los procedimientos TRI y AFI. La tercera investigación, utiliza dos estudios basados en datos simulados tanto para comparar los escalamientos de los sujetos obtenidos por la TRI y la TCT, como para aclarar las condiciones en que es posible esperar la presencia similitudes o diferencias entre ambos...

The Utility of the CVLT-C as an Embedded Validity Indicator After Pediatric Mild Traumatic Brain Injury.

Results of neuropsychological examinations depend on valid data. Whereas clinicians previously believed that clinical skill was sufficient to identify non-credible performance by examinees on standard tests, research demonstrates otherwise. Consequently, studies on measures to detect suspect effort in adults have received tremendous attention in the previous twenty years, and incorporation of validity indicators into neuropsychological examinations is now seen as integral. Few studies exist that validate methods appropriate for the measurement of effort in pediatric populations. Of extant studies, most evaluate standalone measures originally developed for use with adults. The present study examined the utility of indices from the California Verbal Learning Test – Children's Version (CVLT-C) as embedded validity indicators in a pediatric sample. Participants were 225 outpatients aged 8 to 16 years old referred for clinical assessment after mild traumatic brain injury (mTBI). Non-credible performance (n = 39) was defined as failure of the Medical Symptom Validity Test (MSVT). Logistic regression demonstrated that only the Recognition Discriminability index was predictive of MSVT failure (OR = 2.88, p < .001). A cutoff of z ≤ -1.0 was associated with sensitivity of 51% and specificity of 91%. In the current study, CVLT-C Recognition Discriminability was useful in the identification of non-credible performance in a sample of relatively high-functioning pediatric outpatients with mTBI. Thus, this index can be added to the short list of embedded validity indicators appropriate for pediatric neuropsychological assessment.

Polarimetric Response of Rice Fields at C-Band: Analysis and Phenology Retrieval

A set of ten RADARSAT-2 images acquired in fully polarimetric mode over a test site with rice fields in Seville, Spain, has been analyzed to extract the main features of the C-band radar backscatter as a function of rice phenology. After observing the evolutions versus phenology of different polarimetric observables and explaining their behavior in terms of scattering mechanisms present in the scene, a simple retrieval approach has been proposed. This algorithm is based on three polarimetric observables and provides estimates from a set of four relevant intervals of phenological stages. The validation against ground data, carried out at parcel level for a set of six stands and up to nine dates per stand, provides a 96% rate of coincidence. Moreover, an equivalent compact-pol retrieval algorithm has been also proposed and validated, providing the same performance at parcel level. In all cases, the inversion is carried out by exploiting a single satellite acquisition, without any other auxiliary information.

Electrochemical reactions of catechol, methylcatechol and dopamine at tetrahedral amorphous carbon (ta-C) thin film electrodes

The electrochemical reactions of dopamine, catechol and methylcatechol were investigated at tetrahedral amorphous carbon (ta-C) thin film electrodes. In order to better understand the reaction mechanisms of these molecules, cyclic voltammetry with varying scan rates was carried out at different pH values in H2SO4 and PBS solutions. The results were compared to the same redox reactions taking place at glassy carbon (GC) electrodes. All three catechols exhibited quasi-reversible behavior with sluggish electron transfer kinetics at the ta-C electrode. At neutral and alkaline pH, rapid coupled homogeneous reactions followed the oxidation of the catechols to the corresponding o-quinones and led to significant deterioration of the electrode response. At acidic pH, the extent of deterioration was considerably lower. All the redox reactions showed significantly faster electron transfer kinetics at the GC electrode and it was less susceptible toward surface passivation. An EC mechanism was observed for the oxidation of dopamine at both ta-C and GC electrodes and the formation of polydopamine was suspected to cause the passivation of the electrodes.

An adaptive algorithm for clustering cumulative probability distribution functions using the Kolmogorov–Smirnov two-sample test

This paper proposes an adaptive algorithm for clustering cumulative probability distribution functions (c.p.d.f.) of a continuous random variable, observed in different populations, into the minimum homogeneous clusters, making no parametric assumptions about the c.p.d.f.’s. The distance function for clustering c.p.d.f.’s that is proposed is based on the Kolmogorov–Smirnov two sample statistic. This test is able to detect differences in position, dispersion or shape of the c.p.d.f.’s. In our context, this statistic allows us to cluster the recorded data with a homogeneity criterion based on the whole distribution of each data set, and to decide whether it is necessary to add more clusters or not. In this sense, the proposed algorithm is adaptive as it automatically increases the number of clusters only as necessary; therefore, there is no need to fix in advance the number of clusters. The output of the algorithm are the common c.p.d.f. of all observed data in the cluster (the centroid) and, for each cluster, the Kolmogorov–Smirnov statistic between the centroid and the most distant c.p.d.f. The proposed algorithm has been used for a large data set of solar global irradiation spectra distributions. The results obtained enable to reduce all the information of more than 270,000 c.p.d.f.’s in only 6 different clusters that correspond to 6 different c.p.d.f.’s.

l bilanciamento tra le libertà economiche e i diritti sociali collettivi operato dalla Corte di Giustizia è un contemperamento tra diritti equiordinati?* = The balance between economic freedoms and collective social rights and actions; Court of Justice is a choice between rights equiordinati? Actions by the Court of Justice. WP C.S.D.L.E. “Massimo D’Antona”.INT – 98/2012

In the Viking and Laval judgments and more recently in the Comm. v. Germany ruling, the Court of Justice applied the proportionality test to collective rights, setting a series of restrictions to the exercise of the right to strike and the right to collective bargaining. The way the ECJ balances the economic freedoms and the social rights is indeed very different from that of the Italian Constitutional Court. Unlike the European Union Treaties, the Italian Constitution recognizes an important role to the right to take collective action which has to be connected with article 3, paragraph 2, consequently the right of strike is more protected than the exercise of economic freedoms.

Evoluzione dell’inventario di radioisotopi rilevanti ai fini del trattato CTBT per il bando dei test nucleari, tramite l’utilizzo del codice di calcolo ORIGEN-ARP

L’oggetto di questa tesi è lo studio dei radionuclidi rilevanti che vengono generati dalla detonazione di ordigni nucleari. L’obiettivo principale è la ricerca di parametri legati alle sostanze radioattive derivanti dalla fissione nucleare che permettano di capire se c’è stato un rilascio non autorizzato di radioisotopi legati all’esplosione di una bomba nucleare. Inoltre dall’inventario dei nuclidi rilevati si vuole cercare di capire che tipo di tecnologia è stata utilizzata per la costruzione dell’ordigno facendo riferimento a specifici rapporti isotopici. Per ricavare il probabile inventario dei radionuclidi derivanti da fissione è stato utilizzato il codice di calcolo Origen-Arp, programma utilizzato per l’analisi isotopica delle sostanze derivanti dalla “combustione” di materiale fissile all’interno di un reattore nucleare. Il lavoro svolto quindi ha riguardato una parte prettamente sperimentale in cui è stato utilizzato il codice di calcolo e una parte più teorica grazie alla quale è stato possibile contestualizzare i risultati trovati.

COCHRAN: Stata module to test for equality of proportions in matched samples (Cochran's Q)

-cochran- performs a test for equality of two or more proportions in matched samples. The chi-squared calculated by -cochran- is known as Cochran's Q (Cochran 1950)