Combining next-generation pyrosequencing with microarray for large scale expression analysis in non-model species

<p>Abstract</p> <p>Background</p> <p>The next generation sequencing technologies provide new options to characterize the transcriptome and to develop affordable tools for functional genomics. We describe here an innovative approach for this purpose and demonstrate its potential also for non-model species.</p> <p>Results</p> <p>The method we developed is based on 454 sequencing of 3' cDNA fragments from a normalized library constructed from pooled RNAs to generate, through <it>de novo </it>reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This "virtual transcriptome" provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in <it>Vitis vinifera </it>as if no other sequence information was available for this species. The microarray designed on the berries' transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes in the same samples.</p> <p>Conclusion</p> <p>This approach provides a powerful method to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.</p

Identification of PARP-1, Histone H1 and SIRT-1 as new regulators of breast cancer-related aromatase promoter I.3/II

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers

Hybrid optical coating design for omnidirectional antireflection purposes

We present a new design for an omnidirectional antireflection coating for the visible spectral range. In contrast to classical designs, it combines homogeneous layers and linear gradient index layers into one hybrid design with a full thickness of approximately 500nm. The coating may be practically produced based on silicon dioxide as low index material and niobium pentoxide as high index material, while intermediate indices may be obtained from corresponding mixtures

Late Quaternary evolution of rivers, lakes and peatlands in northeast Germany reflecting past climatic and human impact – an overview

Die Kenntnis der regionalen Paläohydrologie ist eine wesentliche Grundlage für das Verständnis aktueller Umweltfragen, wie zum Beispiel nach den Gründen von hydrologischen Veränderungen, dem Einfluss von Landnutzungsstrategien und der Wirksamkeit von Renaturierungsvorhaben in Feuchtgebieten. Auch die Interpretation von Modellierungsergebnissen zu den künftigen Einflüssen des Klima- und Landnutzungswandels auf das Gewässersystem kann durch die Einbeziehung (prä-) historischer Analogien verbessert werden. Für das glazial geprägte nordostdeutsche Tiefland wurde eine Übersicht der vorliegenden paläohydrologischen Befunde für den Zeitraum der letzten etwa 20.000 Jahre erarbeitet. Die Entwicklung der Flüsse wurde mit Blick auf die Tal-/Auengenese und das Ablagerungsmilieu, die Veränderung des Tal- und Gerinneverlaufs sowie den Paläoabfluss bzw. das Paläohochwasser betrachtet. Wesentliche genetische Unterschiede bestehen zwischen Alt- (Elster- und Saalekaltzeit) und Jungmoränengebieten (Weichselkaltzeit) sowie zwischen hoch und tief gelegenen Tälern. Letztere sind stark durch Wasserspiegelveränderungen in der Nord- und Ostsee beeinflusst worden. Die Entwicklung der Seen wurde hinsichtlich der Seebildung, die überwiegend eine Folge der spätpleistozänen bis frühholozänen Toteistieftau-Dynamik ist, und der Veränderungen im Ablagerungsmilieu analysiert. Weiterhin standen Seespiegelveränderungen im Fokus, wobei sich hoch variable lokale Befunde mit einigen Übereinstimmungen zeigten. Der Überblick zur Moorentwicklung konzentrierte sich auf hydrogenetische Moorentwicklungsphasen und auf die langfristige Entwicklung des Grundwasserspiegels. Enge Beziehungen zwischen der Entwicklung der Flüsse, Seen und Moore bestanden insbesondere im Spätholozän durch komplexe Vermoorungsprozesse in den großen Flusstälern. Bis in das Spätholozän wurde die regionale Hydrologie überwiegend durch klimatische, geomorphologische und nicht-anthropogene biologische Faktoren gesteuert. Seit dem Spätmittelalter wurde in der Region das Gewässernetz und der Wasserkreislauf im starken Maß durch anthropogene Interventionen beeinflusst (z.B. Aufstau von Flüssen und Seen, Bau von Kanälen und Deichen, Moorkultivierung). In den letzten etwa 50 Jahren haben dann sogar die kurzfristigen anthropogenen Eingriffe, z.B. in Form von Abflussregulierung, Hydromelioration und künstlicher Seebildung, die Wirksamkeit langfristiger klimatischer und geomorphologischer Prozesse übertroffen.researc

A 1500-year multiproxy record of coastal hypoxia from the northern Baltic Sea indicates unprecedented deoxygenation over the 20th century

The anthropogenically forced expansion of coastal hypoxia is a major environmental problem affecting coastal ecosystems and biogeochemical cycles throughout the world. The Baltic Sea is a semi-enclosed shelf sea whose central deep basins have been highly prone to deoxygenation during its Holocene history, as shown previously by numerous paleoenvironmental studies. However, long-term data on past fluctuations in the intensity of hypoxia in the coastal zone of the Baltic Sea are largely lacking, despite the significant role of these areas in retaining nutrients derived from the catchment. Here we present a 1500-year multiproxy record of near-bottom water redox changes from the coastal zone of the northern Baltic Sea, encompassing the climatic phases of the Medieval Climate Anomaly (MCA), the Little Ice Age (LIA), and the Modern Warm Period (MoWP). Our reconstruction shows that although multicentennial climate variability has modulated the depositional conditions and delivery of organic matter (OM) to the basin the modern aggravation of coastal hypoxia is unprecedented and, in addition to gradual changes in the basin configuration, it must have been forced by excess human-induced nutrient loading. Alongside the anthropogenic nutrient input, the progressive deoxygenation since the beginning of the 1900s was fueled by the combined effects of gradual shoaling of the basin and warming climate, which amplified sediment focusing and increased the vulnerability to hypoxia. Importantly, the eutrophication of coastal waters in our study area began decades earlier than previously thought, leading to a marked aggravation of hypoxia in the 1950s. We find no evidence of similar anthropogenic forcing during the MCA. These results have implications for the assessment of reference conditions for coastal water quality. Furthermore, this study highlights the need for combined use of sedimentological, ichnological, and geochemical proxies in order to robustly reconstruct subtle redox shifts especially in dynamic, non-euxinic coastal settings with strong seasonal contrasts in the bottom water quality.Peer reviewe

Extreme value statistics of smooth random Gaussian fields

We consider the Gumbel or extreme value statistics describing the distribution function p_G(x_max) of the maximum values of a random field x within patches of fixed size. We present, for smooth Gaussian random fields in two and three dimensions, an analytical estimate of p_G which is expected to hold in a regime where local maxima of the field are moderately high and weakly clustered. When the patch size becomes sufficiently large, the negative of the logarithm of the cumulative extreme value distribution is simply equal to the average of the Euler Characteristic of the field in the excursion x > x_max inside the patches. The Gumbel statistics therefore represents an interesting alternative probe of the genus as a test of non Gaussianity, e.g. in cosmic microwave background temperature maps or in three-dimensional galaxy catalogs. It can be approximated, except in the remote positive tail, by a negative Weibull type form, converging slowly to the expected Gumbel type form for infinitely large patch size. Convergence is facilitated when large scale correlations are weaker. We compare the analytic predictions to numerical experiments for the case of a scale-free Gaussian field in two dimensions, achieving impressive agreement between approximate theory and measurements. We also discuss the generalization of our formalism to non-Gaussian fields.Comment: 10 pages, 2 figures, accepted for publication in MNRA

BACCardI - a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison

Bartels D, Kespohl S, Albaum S, et al. BACCardI - a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison. Bioinformatics. 2005;21(7):853-859.Summary: We provide the graphical tool BACCardI for the construction of virtual clone maps from standard assembler output files or BLAST based sequence comparisons. This new tool has been applied to numerous genome projects to solve various problems including (a) validation of whole genome shotgun assemblies, (b) support for contig ordering in the finishing phase of a genome project, and (c) intergenome comparison between related strains when only one of the strains has been sequenced and a large insert library is available for the other. The BACCardI software can seamlessly interact with various sequence assembly packages. Motivation: Genomic assemblies generated from sequence information need to be validated by independent methods such as physical maps. The time-consuming task of building physical maps can be circumvented by virtual clone maps derived from read pair information of large insert libraries