Microarray analysis of autoimmune diseases by machine learning procedures

—Microarray-based global gene expression profiling, with the use of sophisticated statistical algorithms is providing new insights into the pathogenesis of autoimmune diseases. We have applied a novel statistical technique for gene selection based on machine learning approaches to analyze microarray expression data gathered from patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS), two autoimmune diseases of unknown genetic origin that share many common features. The methodology included a combination of three data discretization policies, a consensus gene selection method, and a multivariate correlation measurement. A set of 150 genes was found to discriminate SLE and PAPS patients from healthy individuals. Statistical validations demonstrate the relevance of this gene set from an univariate and multivariate perspective. Moreover, functional characterization of these genes identified an interferon-regulated gene signature, consistent with previous reports. It also revealed the existence of other regulatory pathways, including those regulated by PTEN, TNF, and BCL-2, which are altered in SLE and PAPS. Remarkably, a significant number of these genes carry E2F binding motifs in their promoters, projecting a role for E2F in the regulation of autoimmunity.

Análisis biomecánico del segmento de la carrera a pie de la competición en triatlón

El principal objetivo de la presente investigación fue el conocer el perfil de rendimiento técnico de los triatletas, desde un punto de vista biomecánica, en el segmento carrera a pie durante la competición en triatlón. Asimismo, como el genero y el nivel deportivo del triatleta podrían influir en su respuesta motriz durante la competicion. Para ello, se necesitaba desarrollar y validar una técnica experimental que fuera lo suficientemente precisa (validez interna), con una alta fiabilidad y con una gran validez externa (ecologica) debido al entorno de la competicion. La muestra la formaron un total de 64 deportistas: 32 triatletas participantes en la Copa del Mundo de Triatlon de Madrid-2008 (16 hombres y 16 mujeres) y 32 triatletas participantes en el Clasificatorio del Campeonato de Espana Elite (16 hombres y 16 mujeres). El análisis de la técnica de carrera de los deportistas se realizo mediante un sistema fotogramétrico en 2d que permitió calcular las coordenadas (x,y) de los centros articulares con un error de 1.66% en el eje x y de un 2.10% en el eje y. Las imágenes fueron obtenidas por una cámara que filmaba el movimiento en un plano antero-posterior del triatleta. Algoritmos basados en la DLT (Abdel-Aziz & Karara, 1971) permitieron conocer las coordenadas reales a partir de las coordenadas digitalizadas en el plano y posteriormente las distintas variables analizadas. El análisis biomecánica de la carrera se realizo en 4 ocasiones diferentes durante la competición, correspondiendo con cada una de las vueltas de 2,5 km, que el triatleta tenía que realizar. La velocidad de carrera resulto estar íntimamente ligada al nivel deportivo del triatleta. Del mismo modo, 3 de los 4 grupos analizados presentaron valores inferiores a 3 minutos 30 segundos por kilometro recorrido, poniendo de manifiesto el altísimo nivel de los sujetos analizados. Del mismo modo parece que las chicos consiguen una mayor velocidad gracias a una mayor longitud de ciclo en relación a las chicas, ya que estas muestran valores mayores en cuanto a frecuencia de zancada. La frecuencia de zancada presento los valores más altos en la primera vuelta en todos los deportistas analizados. Asimismo, los triatletas de nivel internacional y las chicas fueron los que mostraron los mayores valores. La longitud de zancada presento distintas tendencias en función del nivel y el género del deportista. Así pues, en los deportistas internacionales y en los chicos los mayores valores se encontraron en la primera vuelta mientras que la tendencia fue al descenso, siendo probablemente la fatiga acumulada la causante de dicha tendencia. En cambio, aquellos deportistas de nivel nacional y las chicas mostraron valores mayores en la segunda vuelta que en la primera, evidenciando que además de la fatiga, el ciclismo previo tiene una incidencia directa sobre su rendimiento. Los tiempos de vuelo permanecieron constantes durante toda la carrera, encontrando cierta evolución en los tiempos de apoyo, la cual provoca una modificación en los porcentajes relativos en los tiempos de vuelo. Los tiempos de apoyo más bajos se encontraron en la primera vuelta. Del mismo modo, los deportistas de nivel internacional y los chicos mostraron valores inferiores. También, estos grupos fueron más constantes en sus valores a lo largo de las vueltas. Por el contrario, se encontraron tendencias al aumento en los triatletas de nivel nacional y en las chicas, los cuales no fueron capaces de mantener el mismo rendimiento debido seguramente a su menor nivel deportivo. La oscilación vertical de la cadera se mostro constante en los triatletas de mayor nivel, encontrándose tendencias al aumento en los de menor nivel. Del mismo modo, los valores más altos correspondieron a las chicas y a los deportistas de nivel nacional. La distancia de la cadera al apoyo permaneció constante a lo largo de las vueltas en todos los grupos, obteniéndose valores mayores en los triatletas de nivel internacional y en los chicos. El ángulo de la rodilla apoyada en el momento del despegue no mostro una tendencia clara. Los deportistas de nivel internacional y los chicos presentaron los valores más bajos. El ángulo de la rodilla libre en el momento del despegue mostro una correlación muy alta con la velocidad de carrera. Del mismo modo, los ángulos más pequeños se encontraron en los triatletas internacionales y en los chicos, debido seguramente a los mayores valores de velocidad registrados por ambos grupos. Los ángulos de los tobillos no mostraron ninguna tendencia clara durante la competición analizada. Los cuatro grupos de población presentaron valores similares, por lo que parece que no representan una variable que pueda incidir sobre el rendimiento biomecánica del triatleta. Los resultados obtenidos en el presente estudio de investigación avalan la utilización de la fotogrametría-video en 2d para el análisis de la técnica de carrera durante la competición en triatlón. Su aplicación en una competición de máximo nivel internacional ha posibilitado conocer el perfil técnico que presentan los triatletas a lo largo del segmento de carrera a pie. Del mismo modo, se ha podido demostrar como los estudios realizados en laboratorio no reflejan la realidad competitiva de un triatlón de máximo nivel. The aim of this research was to determine the running technique profile during a triathlon competition from a biomechanical perspective. Also, to analyze the triathlete gender’s and level of performance’s influence on this profile in competition. An accurate (internal validity) and reliable methodology with a high external validity (ecological) had to be developed to get those aims in competition. Sixty-four triathletes were analyzed. 32 (16 males, 16 females) took part in the Madrid 2008 Triathlon World Cup and 32 (16 males and 16 females) took part in the Spanish Triathlon National Championships. The biomechanical analyses were carried out by a photogrammetric system that allow to calculate the landmarks coordinates (x,y) with a 1.66% error in x axis, and a 2.10% error in y axis. The frames were obtained with a camera situated perpendicular to the triathletes’ trajectory, filming the saggittal plane. DLT based algorithms (Abdel-Aziz & Karara, 1971) were used to calculate the real coordinates from the digitalized ones and the final variables afterwards. The biomechanical analisys itself was performed in four different moments during the competition, according to each 2.5 km lap the triathletes had to do. Running speed was highly related to performance level. Also, 3 of the 4 analyzed groups showed speed values under the 3 minutes and 30 seconds per kilometer. It demonstrated the very high performance level of the analized triathletes. Furthermore, it seems that men get higher speeds because their longer stride length, while women shows higher stride frequency values. The highest stride frequency values were found in the first lap. Women and the international level triathletes showed the highest values. Stride length showed different tendencies according to the gender and level of performance. Men and international level triathletes showed the highest level in the first lap and a decreasing tendency after that. The accumulated fatigue was probably the reason of this tendency. On the other hand, higher values than in first lap were found in the second one in women and national level triathletes. It demonstrated the previous cycling can affect to those groups in terms of biomechanics. Flight times remained constant during the running part, while the contact times showed an increasing tendency that caused a variation in flight times percents. The lowest contact times were found in the first lap and in men and international triathletes’ values. Also, these two groups were more consistent during the whole running. On the other hand, increasing tendencies were found in women and national level triathletes, who were not able to maintain the same values probably due to their lower level of performance. Higher level triathletes showed more consistent hip vertical oscillation values than lower level triathletes, who presented increasing tendencies. The highest values were found in women and national level triathletes. The horizontal distance hip-toe cap remained constant among the laps in all the groups. Men and international level triathletes showed the highest values. The support knee angle at toe-off did not show a clear tendency. The lowest values were found in men and international level triathletes. A high correlation was found between the non-support knee angle and the running speed. Furthermore, men and international level triathletes showed the smallest values, due to the higher velocities reached by these two groups. Ankles angles did not show any tendency during the running part. Similar values were found in the four analyzed groups, so this variable does not seem to represent an important one within the triathlete’s performance. The results obtained in the present research support the use of the bidimensional photogrammetric video-system to analyze the running technique during a triathlon competition. Its application in international triathlon meetings has allowed determining the triathletes’ technique profile during the running part. Also, it has been demonstrated the laboratory-based studies does not reproduce a top-level competition.

Self-similar motion of laser half-space plasmas. I. Deflagration regime

The one-dimensional self-similar motion of an initially cold, half-space plasma of electron density 0,produced by the (anomalous) absorption of a laser pulse of irradiation = (j>0f/T(0< (< T) at the critical density nc(«c/«0=exchange and retains three dimensionless numbers: e, Zt (ion charge number), and a = (9/c/4m,) (T/C 2n l/4>oKe)213, where k, m, are Boltzmann's constant and the ion mass, and Ke X (electron temperature)5'2 = heat conductivity. If a >e- 4 ' 3 , a deflagration wave separates an isentropic compression with a shock bounding the undisturbed plasma, and an isentropic expansion flow to the vacuum. The structures of these three regions are completely determined; in particular, the Chapman-Jouguet condition is proved and the density behind the deflagration is found. The deflagration-compression thickness ratio is large (small) for a^e- 5 ' 3(a>e- 5 ' 3 ) . The compression to expansion ratio for both energy and thickness is 0(e"2). For Z,- large, a deflagration exists even if a~e~413. Condition a>e~4'3 may be applied to pulses that are not linear.

Efecto del cereal base y del programa de alimetnación sobre los parámetros productivos en pollitas de 1 a 17 semanas de edad.

Maíz y trigo son los cereales más utilizados en piensos para aves. El maíz tiene menos proteína (7,7 vs. 11,2%) pero más energía (3.260 vs. 3.150 Kcal EMAn/kg) que el trigo (FEDNA, 2010). Además, el valor nutricional es más variable para el trigo que para el maíz Kim et al., 1976; Mollah et al., 1983). En pollos y en ponedoras, numerosos trabajos indican que el trigo puede ser utilizado en sustitución del maíz como ingrediente principal del pienso Mathlouthi et al., 2002; Carré et al., 2002; Lázaro et al., 2003; Pérez-Bonilla et al., 2011). Frikha et al. (2009), observaron que las pollitas alimentadas con maíz tenían mayores GMD pero IC similares que las pollitas alimentadas con trigo. La presentación del pienso afecta los rendimientos productivos en avicultura. Amerah et al. (2007) mostraron un aumento de la GMD y una mejora del IC en pollos alimentados con gránulos en comparación con los alimentados con harina. Gous y Morris (2001) observaron que pollitas alimentadas con migas de 1 a 4 sem y con gránulo de 5 a 20 sem de edad, consumieron 2% menos pienso pero pesaron un 6% más que las alimentadas con harina durante toda la recría. Frikha et al. (2009) observaron un incremento en la GMD en las pollitas alimentadas con gránulo respecto a las pollitas alimentadas con harina. Los autores no han encontrado información alguna sobre el efecto de alternar la presentación del pienso (bien en harina, bien en miga) durante las distintas etapas del periodo de recría (0-5, 5-10 y 10-17 sem de vida) sobre la productividad de las aves. El presente trabajo estudió la influencia del cereal base del pienso y de cambios en la presentación del mismo durante las 3 fases del programa de alimentación en recría sobre los parámetros productivos en pollitas de 1 a 17 semanas de edad.

Feed form and energy concentration of the diet affect growth performance and digestive tract traits of brown-egg laying pullets from hatching to 17 weeks of age

The influence of feed form and energy concentration of the diet on growth performance and the development of the gastrointestinal tract (GIT) was studied in brown-egg laying pullets. Diets formed a 2 x 5 factorial with 2 feed forms (mash vs. crumbles) and 5 levels of energy differing in 50 kcal AMEn/kg. For the entire study (0 to 17 wk of age) feeding crumbles increased ADFI (52.9 vs. 49.7 g; P < 0.001) and ADG (12.7 vs. 11.6 g; P < 0.001) and improved feed conversion ratio (FCR; 4.18 vs. 4.27; P < 0.001). An increase in the energy content of the diet decreased ADFI linearly (P < 0.001) and improved FCR quadratically (P < 0.01) but energy intake (kcal AMEn/d) was not affected. BW uniformity was higher (P < 0.05) in pullets fed crumbles than in those fed mash but was not affected (P > 0.05) by energy content of the diet. At 5, 10, and 17 wk of age, the relative weight (RW, % BW) of the GIT and the gizzard, and gizzard digesta content were lower (P < 0.05 to P < 0.001) and gizzard pH was higher (P < 0.05 to P < 0.001) in pullets fed crumbles than in pullets fed mash. Energy concentration of the diet did not affect any of the GIT variables studied. In summary, feeding crumbles improved pullet performance and reduced the RW of the GIT and gizzard, and increased gizzard pH at all ages. An increase in the energy content of the diet improved FCR from 0 to 17 wk of age. The use of crumbles and the increase in the AMEn content of the diet might be used adventageously when the objetive is to increase the BW of the pullets. However, crumbles affected the development and weight of the organs of the GIT, which might have negative effects on feed intake and egg production at the beginning of the egg laying cycle.

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21WAF1/Cip-1/Sdi and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21WAF1/Cip-1/Sdi protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-loxP-mediated gene inversion is used to change the specificity of the BCR on mature IgM+ IgD+ B cells in vivo to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive bcl-2 transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.