570 Biowissenschaften; Biologie
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- Circus pygargus (1)
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Technische Prozesse können erheblich durch Biofilme beeinflußt werden und spielen in weiten Bereichen der Technik und Biotechnologie eine große Rolle. Ein zentrales Problem beim Einsatz von Werkstoffen in biologischen Systemen ist die Ansiedlung von Zellen und deren Stoffwechselprodukten auf Festkörperoberflächen. Erst hiervon gehen alle nachfolgenden Reaktionen aus, wie Kontamination von Implantaten und medizinischen Geräten, Biokorrosion und die Bildung von Biofilmen in technischen Geräten.
Die in der Biotechnologie eingesetzten Werkstoffe müssen bestimmten technischen und biologischen Forderungen genügen. Die Klärung der Adhäsionsmechanismen der Bakterien auf unterschiedlichen Werkstoffoberflächen und des sich aus einem ersten Zellhaufen entwickelnden Biofilms sowie die Reaktionen und die Transportmechanismen an und in ihm sind die Voraussetzung zur Interpretation der Werkstoffschädigung und des Zellverhaltens. Neben der konventionellen Licht- und Rasterelektronenmikroskopie steht mit der Raster-Kraft- Mikroskopie (SFM) ein neues Verfahren zur Abbildung von Oberflächen biologischer Systeme mit hoher Auflösung zur Verfügung. Ziel der Arbeiten ist es, mit dem SFM Untersuchungen von Biofilmen auf verschiedenen Werkstoffoberflächen durchzuführen. Hierzu ist es notwendig, die Raster-Kraft-Mikroskopie an die Besonderheiten biologischer Strukturen anzupassen. Aufgrund dieser Oberflächenuntersuchungen lassen sich dann geeignete Werkstoffe und Beschichtungen für den Einsatz in biologischen Systemen auswählen.
We have re-analyzed previously published gene expression data from ninety-four pancreatic ductal adenocarcinomas (PDAC) samples. We determined the gene expression profile of genes differentially expressed in PDAC compared to non-malignant pancreatic tissue. Using the 100 top-ranked genes, we were able to discriminate between PDAC and non-malignant pancreatic tissue. A hierarchical cluster analysis revealed only a 6 % false discovery rate. The prognostic strength of these discriminative genes was underscored by a SVM classification and 3-fold cross validation with an 89 % correct class assignment. The annotation of the 100 top-ranked genes revealed that most of the genes were involved in the processes of signal transduction, cell adhesion, extracellular matrix organization and cell migration. The most greatly affected signal cascade was the transforming growth factor β receptor signaling pathway, which was significantly enriched in the top-ranked genes. Furthermore, we identified eleven genes that were associated with good prognosis.
The SYSTERS project aims to provide a meaningful partitioning of the whole protein sequence space by a fully automatic procedure. A refined two-step algorithm assigns each protein to a family and a superfamily. The sequence data underlying SYSTERS release 4 now comprise several protein sequence databases derived from completely sequenced genomes (ENSEMBL, TAIR, SGD and GeneDB), in addition to the comprehensive Swiss-Prot/TrEMBL databases. The SYSTERS web server (http://systers.molgen.mpg.de) provides access to 158 153 SYSTERS protein families. To augment the automatically derived results, information from external databases like Pfam and Gene Ontology are added to the web server. Furthermore, users can retrieve pre-processed analyses of families like multiple alignments and phylogenetic trees. New query options comprise a batch retrieval tool for functional inference about families based on automatic keyword extraction from sequence annotations. A new access point, PhyloMatrix, allows the retrieval of phylogenetic profiles of SYSTERS families across organisms with completely sequenced genomes.
Background
Tardigrades represent an animal phylum with extraordinary resistance to environmental stress.
Results
To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data.
Conclusion
Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.
mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.
The use of a priori knowledge in the alignment of targeted sequencing data is investigated using computational experiments. Adapting a Needleman–Wunsch algorithm to incorporate the genomic position information from the targeted capture, we demonstrate that alignment can be done to just the target region of interest. When in addition use is made of direct string comparison, an improvement of up to a factor of 8 in alignment speed compared to the fastest conventional aligner (Bowtie) is obtained. This results in a total alignment time in targeted sequencing of around 7 min for aligning approximately 56 million captured reads. For conventional aligners such as Bowtie, BWA or MAQ, alignment to just the target region is not feasible as experiments show that this leads to an additional 88% SNP calls, the vast majority of which are false positives (∼92%).
Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.
In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.
DNA methylation is a widely conserved epigenetic modification. The analysis of genome-scale DNA methylation patterns in various organisms suggests that major features of animal methylomes are widely conserved. However, based on the variation of DNA methyltransferase genes in invertebrates, it has also been proposed that DNA methylation could provide a molecular mechanism for ecological adaptation. We have now analyzed the methylome of the desert locust, Schistocerca gregaria, which represents an organism with a high degree of phenotypic plasticity. Using genome-scale bisulfite sequencing, we show here that the S. gregaria methylome is characterized by CpG- and exon-specific methylation and thus shares two major features with other animal methylomes. In contrast to other invertebrates, however, overall methylation levels were substantially higher and a significant fraction of transposons was methylated. Additionally, genic sequences were densely methylated in a pronounced bimodal pattern, suggesting a role for DNA methylation in the regulation of locust gene expression. Our results thus uncover a unique pattern of genome methylation in locusts and provide an important foundation for investigating the role of DNA methylation in locust phase polyphenism.