The role of AKT-mediated phosphorylation in suppression of MAD1 and the development of new approach in protein complex detection

MAX dimerization protein 1 (MAD1) is a basic-helix-loop-helix transcription factors that recruits transcription repressor such as HDAC to suppress target genes transcription. It antagonizes to MYC because the promoter binding sites for MYC are usually also serve as the binding sites for MAD1 so they compete for it. However, the mechanism of the switch between MYC and MAD1 in turning on and off of genes' transcription is obscure. In this study, we demonstrated that AKT-mediated MAD1 phosphorylation inhibits MAD1 transcription repression function. The association between MAD1 and its target genes' promoter is reduced after been phosphorylated by AKT; therefore, consequently, allows MYC to occupy the binding site and activates transcription. Mutation of such phosphorylation site abrogates the inhibition from AKT. In addition, functional assays demonstrated that AKT suppressed MAD1-mediated transcription repression of its target genes hTERT and ODC. Cell cycle and cell growth were also been released from inhibition by MAD1 in the presents of AKT. Taken together, our study suggests that MAD1 is a novel substrate of AKT and AKT-mediated MAD1 phosphorylation inhibits MAD1function; therefore, activates MAD1 target genes expression. ^ Furthermore, analysis of protein-protein interaction is indispensable for current molecular biology research, but multiplex protein dynamics in cells is too complicated to be analyzed by using existing biochemical methods. To overcome the disadvantage, we have developed a single molecule level detection system with nanofluidic chip. Single molecule was analyzed based on their fluorescent profile and their profiles were plotted into 2 dimensional time co-incident photon burst diagram (2DTP). From this 2DTP, protein complexes were characterized. These results demonstrate that the nanochannel protein detection system is a promising tool for future molecular biology. ^

Identification of genetic risk factors for cerebellar mutism in pediatric brain tumor patients

Following posterior fossa surgery for resection of childhood medulloblastoma and primitive neuroectodermal tumor (M/PNET), cerebellar mutism (CM) may develop. This is a condition of absent or diminished speech in a conscious patient with no evidence of oral apraxia, which can be accompanied by other symptoms of the posterior fossa syndrome complex, which includes ataxia and hypotonia. Little is known about the etiology. Therefore, we conducted a SNP, gene, and pathway-level analysis to assess the role of host genetic variation on the risk of CM in M/PNET subjects following treatment. Cases (n= 20) and controls (n= 53) were recruited from the Childhood Cancer Epidemiology and Prevention Center, in Houston, TX. DNA samples were genotyped using the Illumina Human 1M Quad SNP chip. Ten pathways were identified from logistic regression used to identify the marginal effect of each SNP on CM risk. The minP test was conducted to identify associations between SNPs categorized to genes and CM risk. Pathways were assessed to determine if there was a significant enrichment of genes in the pathway compared to all other pathways. There were 78 genes that reached the threshold of min P ≤0.05 in 948 genes. The Neurotoxicity pathway was the most significant pathway after adjusting for multiple comparisons (q=0.040 and q=0.005, using Fisher's exact test and a test of proportions, respectively). Most genes within the Neurotoxicity pathway that reached a threshold of minP ≤0.05 were known to have an apoptosis function, possibly inducing neuronal apoptosis in the dentatothalamocortical pathway, and may be important in CM etiology in this population. This is the first study to assess the potential role of genetic risk factors on CM. As an exploratory study, these results should be replicated in a larger sample. ^

Galectin-3 Enhances the Malignant Melanoma Phenotype by Regulating Autotaxin

In melanoma patient specimens and cell lines, the over expression of galectin-3 is associated with disease progression and metastatic potential. Herein, we have sought out to determine whether galectin-3 affects the malignant melanoma phenotype by regulating downstream target genes. To that end, galectin-3 was stably silenced by utilizing the lentivirus-incorporated small hairpin RNA in two metastatic melanoma cell lines, WM2664 and A375SM, and subjected to gene expression microarray analysis. We identified and validated the lysophospholipase D enzyme, autotaxin, a promoter of migration, invasion, and tumorigenesis, to be down regulated after silencing galectin-3. Silencing galectin-3 significantly reduced the promoter activity of autotaxin. Interestingly, we also found the transcription factor NFAT1 to have reduced protein expression after silencing galectin-3. Electrophoretic mobility shift assays from previous reports have shown that NFAT1 binds to the autotaxin promoter in two locations. ChIP analysis was performed, and we observed a complete loss of bound NFAT1 to the autotaxin promoter after silencing galectin-3 in melanoma cells. Mutation of the NFAT1 binding sites at either location reduces autotaxin promoter activity. Silencing NFAT1 reduces autotaxin expression while over expressing NFAT1 in NFAT1 negative SB-2 melanoma cells induces autotaxin expression. These data suggest that galectin-3 silencing reduces autotaxin transcription by reducing the amount of NFAT1 protein expression. Rescue of galectin-3 rescues both NFAT1 and autotaxin. We also show that the re-expression of autotaxin in galectin-3 shRNA melanoma cells rescues the angiogenic phenotype in vivo. Furthermore, we identify NFAT1 as a potent inducer of tumor growth and experimental lung metastasis. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin and assign a novel role for NFAT1 during melanoma progression.

GENOME-WIDE PROFILING UNVEILS CRITICIAL FUNCTIONS OF p53 IN HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) possess two unique characteristics: infinite self-renewal and the potential to differentiate into almost every cell type (pluripotency). Recently, global expression analyses of metastatic breast and lung cancers revealed an ESC-like expression program or signature, specifically for cancers that are mutant for p53 function. Surprisingly, although p53 is widely recognized as the guardian of the genome, due to its roles in cell cycle checkpoints, programmed cell death or senescence, relatively little is known about p53 functions in normal cells, especially in ESCs. My hypothesis is that p53 has specific transcription regulatory functions in human ESCs (hESCs) that a) oppose pluripotency and b) protect the stem cell genome in response to DNA damage and stress signaling. In mouse ESCs, these roles are believed to coincide, as p53 promotes differentiation in response to DNA damage, but this is unexplored in hESCs. To determine the biological roles of p53, specifically in hESCs, we mapped genome-wide chromatin interactions of p53 by chromatin immunoprecipitation and massively parallel tag sequencing (ChIP-Seq), and did so under three VIdifferent conditions of hESC status: pluripotency, differentiation-initiated and DNA-damage-induced. ChIP-Seq showed that p53 is enriched at distinct, induction-specific gene loci during each of these different conditions. Microarray gene expression analysis and functional annotation of the distinct p53-target genes revealed that p53 regulates specific genes encoding developmental regulators, which are expressed in differentiation-initiated but not DNA- damaged hESCs. We further discovered that, in response to differentiation signaling, p53 binds regions of chromatin that are repressed but also poised for rapid activation by core pluripotency factors OCT4 and NANOG in pluripotent hESCs. In response to DNA damage, genes associated with migration and motility are targeted by p53; whereas, the prime targets of p53 in control of cell death are conserved for p53 regulation in both differentiation and DNA damage. Our genome-wide profiling and bioinformatics analyses show that p53 occupies a special set of developmental regulatory genes during early differentiation of hESCs and functions in an induction-specific manner. In conclusion, our research unveiled previously unknown functions of p53 in ESC biology, which augments our understanding of one of the most deregulated proteins in human cancers.

Grandparental immune priming in S.typhle - Gene expression and Size of F2

Data table shows life history (size-length) and gene expression measurements of 44 target genes and 4 housekepping genes for 192 samples (F2 juveniles) of the experiment "Grandparental immune priming in Syngnathus typhle". Gene expression was measured using Fluidigm chip systems in May 2014. Shown are the mean Ct values (Cycle time) of two technical replicates.

Modelización de la distribución espacial de Bactericera cockerelli Sulc. (Hemiptera: Triozidae) en Solanum tuberosum L. (Solanales: Solanaceae)

La producción de Solanum tuberosum L., Lycopersicum esculentum Mill. y Physalis ixocarpa Brot. (Solanales: Solanaceae) ha sufrido fuertes pérdidas económicas por la presencia de Bactericera cockerelli Sulc. (Hemiptera: Triozidae) al asociarse con las enfermedades punta morada o “zebra chip", además de ser el transmisor de Candidatus Liberibacter solanacearum. Las alternativas de control utilizadas han carecido de eficacia por desconocer la distribución espacial del insecto dentro de la parcela. Conocer dicho comportamiento permitiría focalizar las alternativas de control, haciéndolas más eficaces. Este trabajo tuvo por objetivo modelizar la distribución espacial de los estadíos de huevo, ninfa y adulto de B. cockerelli obtenidos en muestreos por transectos en un cultivo de papa, utilizando herramientas geoestadísticas. Los resultados indican que la distribución espacial de las poblaciones de huevos, ninfas y adultos de B. cockerelli fue de tipo agregada en cada fecha de muestreo. La validación cruzada de los semivariogramas obtenidos corrobora la distribución agregada en las poblaciones de B. cockerelli. Por su parte, los mapas elaborados permiten observar la estructura agregada de las poblaciones del insecto, permitiendo identificar áreas infestadas y áreas libres. Se encontró estabilidad espacio temporal para los tres estadios del insecto.

EPICA Dronning Maud Land EDML ice core drilling protocol

We report on the EPICA Dronning Maud Land (East Antarctica) deep drilling operation. Starting with the scientific questions that led to the outline of the EPICA project, we introduce the setting of sister drillings at NorthGRIP and EPICA Dome C within the European ice-coring community. The progress of the drilling operation is described within the context of three parallel, deep-drilling operations, the problems that occurred and the solutions we developed. Modified procedures are described, such as the monitoring of penetration rate via cable weight rather than motor torque, and modifications to the system (e.g. closing the openings at the lower end of the outer barrel to reduce the risk of immersing the drill in highly concentrated chip suspension). Parameters of the drilling (e.g. core-break force, cutter pitch, chips balance, liquid level, core production rate and piece number) are discussed. We also review the operational mode, particularly in the context of achieved core length and piece length, which have to be optimized for drilling efficiency and core quality respectively. We conclude with recommendations addressing the design of the chip-collection openings and strictly limiting the cable-load drop with respect to the load at the start of the run.

Strontium and rubidium isotopes in serpentinites from ODP Site 195-1200

Subduction of the Pacific plate beneath the Mariana forearc releases fluids to the overlying mantle wedge that ascend, producing serpentinite "mud" that discharges on the ocean floor. As part of Leg 195 of the Ocean Drilling Program cores were obtained from drill-holes into the mud volcanoes. We report the isotopic composition of Sr in water squeezed from intervals of the cores, in the serpentinite mud, in leaches of the serpentinite mud, and in entrained small harzburgitic clasts. Except in the upper few meters below the seawater-mud interface, where pore water approaches seawater Sr concentration and isotopic ratio, Sr concentration and isotopic composition remain constant at 3-6 µmol/kg and ~0.7054. Because the elemental chemistry of the pore water is unlike seawater, this isotopic composition reflects fluids derived from the subducted slab, probably modified by reaction with mantle material during ascent. Higher Sr isotopic ratios, up to 0.7087, - but not with higher Sr concentrations in pore water - occur superimposed on an advection profile at 13-16 mbsf surrounding a thin layer of foraminiferal sand. Since the upward seepage velocity of slab fluids in the mud volcano vents is a few cm/yr, exchange of Sr between these carbonates and the rising fluids must have occurred within a maximum of a few hundred years, essentially instantaneously given the millions, or tens of millions, of years the mud volcanoes have been in existence. In contrast, the strontium isotopic compositions of leached serpentinite mud, and of small harzburgite clasts entrained in the mud, are always significantly greater than that of the pore water. In small harzburgite clasts the ratio reaches 0.7088, almost as high as the seawater value of 0.7092 and much higher than the value of typical mantle-derived strontium of ~0.704. The serpentinite muds and harzburgite clasts clearly equilibrated with seawater Sr when they were initially deposited at the surface of the seamount, but following burial they have not fully equilibrated with strontium in the pore water now discharging through the vents. These variations in the strontium isotopic composition of solids and pore waters are more consistent with episodic expulsion of fluids in the subduction zone than steady state flow. Whereas strontium in carbonates equilibrates isotopically within a few hundred years, strontium in buried harzburgite clasts does not equilibrate in the same time, assuming steady state rates of upward fluid flow. By inference, the harzburgite clasts and associated serpentinite mud must have been near the seafloor, unburied, for a yet undetermined but much longer period of time to have equilibrated from ~0.704 to 0.709 prior to subsequent burial. It may be possible to characterize at least the periodicity of fluid release in the mud volcano setting by investigating the zonation of strontium isotopic composition of hartzburgite clasts throughout the 60-meter deep composite cores.

Pleistocene chronology of sediments from the Lomonosov Ridge

Despite its importance in the global climate system, age-calibrated marine geologic records reflecting the evolution of glacial cycles through the Pleistocene are largely absent from the central Arctic Ocean. This is especially true for sediments older than 200 ka. Three sites cored during the Integrated Ocean Drilling Program's Expedition 302, the Arctic Coring Expedition (ACEX), provide a 27 m continuous sedimentary section from the Lomonosov Ridge in the central Arctic Ocean. Two key biostratigraphic datums and constraints from the magnetic inclination data are used to anchor the chronology of these sediments back to the base of the Cobb Mountain subchron (1215 ka). Beyond 1215 ka, two best fitting geomagnetic models are used to investigate the nature of cyclostratigraphic change. Within this chronology we show that bulk and mineral magnetic properties of the sediments vary on predicted Milankovitch frequencies. These cyclic variations record "glacial" and "interglacial" modes of sediment deposition on the Lomonosov Ridge as evident in studies of ice-rafted debris and stable isotopic and faunal assemblages for the last two glacial cycles and were used to tune the age model. Potential errors, which largely arise from uncertainties in the nature of downhole paleomagnetic variability, and the choice of a tuning target are handled by defining an error envelope that is based on the best fitting cyclostratigraphic and geomagnetic solutions.

Biogeochemical measurements associated to deep-sea wood falls

Large organic food falls to the deep sea - such as whale carcasses and wood logs - support the development of reduced, sulfidic niches in an otherwise oxygenated, oligotrophic deep-sea environment. These transient hot spot ecosystems may serve the dispersal of highly adapted chemosynthetic organisms such as thiotrophic bivalves and siboglinid worms. Here we investigated the biogeochemical and microbiological processes leading to the development of sulfidic niches. Wood colonization experiments were carried out for the duration of one year in the vicinity of a cold seep area in the Nile deep-sea fan (Eastern Mediterranean) at depths of 1690 m. Wood logs were deployed in 2006 during the BIONIL cruise (RV Meteor M70/2 with ROV Quest, Marum, Germany) and sampled in 2007 during the Medeco-2 cruise (RV Pourquoi Pas? with ROV Victor 6000, Ifremer, France). Wood-boring bivalves played a key role in the initial degradation of the wood, the dispersal of wood chips and fecal matter around the wood log, and the provision of colonization surfaces to other organisms. Total oxygen uptake measured with a ROV-operated benthic chamber module was higher at the wood (0.5 m away) in contrast to 10 m away at a reference site (25 mmol m-2 d-1 and 1 mmol m-2 d-1, respectively), indicating an increased activity of sedimentary communities around the wood falls. Bacterial cell numbers associated with wood increased substantially from freshly submerged wood to the wood chip/fecal matter layer next to the wood experiments, as determined with Acridine Orange Direct Counts (AODC) and DAPI-stained counts. Microsensor measurements of sulfide, oxygen and pH were conducted ex situ. Sulfide fluxes were higher at the wood experiments when compared to reference measurements (19 and 32 mmol m-2 d-1 vs. 0 and 16 mmol -2 d-1, respectively). Sulfate reduction (SR) rates at the wood experiments were determined in ex situ incubations (1.3 and 2.0 mmol m-2 d-1) and fell into the lower range of SR rates previously observed from other chemosynthetic habitats at cold seeps. There was no influence of wood deposition on phosphate, silicate and nitrate concentrations, but ammonium concentrations were elevated at the wood chip-sediment boundary layer. Concentrations of dissolved organic carbon were much higher at the wood experiments (wood chip-sediment boundary layer) in comparison to measurements at the reference sites, which may indicate that cellulose degradation was highest under anoxic conditions and hence enabled by anaerobic benthic bacteria, e.g. fermenters and sulfate reducers. Our observations demonstrate that, after one year, the presence of wood at the seafloor had led to the creation of sulfidic niches, comparable to what has been observed at whale falls, albeit at lower rates.

Transporte dependiente de espín en estructuras de magnetorresistencia gigante con capas delgadas de gadolinio

La investigación realizada en este trabajo de tesis se ha centrado en la caracterización y optimización del sistema Fe/Gd/Fe y en el estudio de su efecto en el transporte dependiente de espín y en la transferencia de espín (STT) en dispositivos magnéticos. El fenómeno de STT, uno de los grandes descubrimientos de la espintrónica, se basa en la transferencia de momento angular de una corriente polarizada de espín a la imanación local de un material magnético. Este efecto se traduce en que una corriente polarizada de espín puede provocar variaciones en la imanación del material sin necesidad de campo magnético aplicado. Este fenómeno necesita una densidad de corriente muy alta, de manera que sus efectos solo se observan en dispositivos de tamaño nanométrico a partir de la llamada densidad de corriente crítica. El STT tiene un gran potencial tecnológico para distintas aplicaciones, como emisores de radiofrecuencia para comunicación in-chip o memorias magnéticas alternativas, en que se podría leer y escribir la información únicamente mediante corriente, sin necesidad de aplicar campo magnético ni utilizar bobinas de detección. Desde el punto de vista de este tipo de aplicaciones hay un gran interés en disminuir la densidad de corriente crítica a la que empieza a observarse el efecto. Sin embargo, hay otro tipo de dispositivos en que el STT supone un problema o factor limitante. Este es el caso de las cabezas lectoras de ordenador, en las que a partir de la densidad de corriente crítica aparece ruido e inestabilidad adicional en la señal inducidos por STT, lo que limita su sensibilidad. Para este tipo de aplicación, se desea por tanto que la corriente crítica a partir de la cual aparece ruido e inestabilidad adicional en la señal sea tan grande como sea posible. El Gd (y especialmente el sistema Fe/Gd/Fe) tiene unas características muy particulares con potencial para afectar varias propiedades relacionadas con la densidad de corriente crítica de un dispositivo de STT. Por este motivo, resulta interesante introducir la tricapa Fe/Gd/Fe en la capa libre de este tipo de dispositivos y estudiar cómo afecta a su estabilidad. Para ello, una primera parte del trabajo se ha centrado en la exhaustiva caracterización del sistema Fe/Gd/Fe y la optimización de sus propiedades de cara a su introducción en la capa libre de dispositivos de STT. Por otro lado, la intención final es alterar o controlar el efecto de transferencia de espín en un dispositivo afectando lo menos posible al resto de las propiedades intrínsecas de su funcionamiento (por ejemplo, al valor de su magnetorresistencia). Por tanto, ha sido necesario estudiar los efectos del sistema Fe/Gd/Fe en el transporte de espín y determinar la manera de introducir la tricapa en el dispositivo optimizando el resto de sus propiedades o afectándolas lo menos posible. Finalmente, hemos introducido el sistema Fe/Gd/Fe en la capa libre de nanodispositivos y hemos estudiado su efecto en la corriente crítica de inestabilidad por STT. Los resultados muestran que estas tricapas Fe/Gd/Fe pueden suponer una solución potencial para los problemas de estabilidad de algunos nanodispositivos magnéticos como las cabezas lectoras magnéticas.