Daniela Beisser

BioNet: an R-Package for the functional analysis of biological networks

MOTIVATION Increasing quantity and quality of data in transcriptomics and interactomics create the need for integrative approaches to network analysis. Here, we present a comprehensive R-package for the analysis of biological networks including an exact and a heuristic approach to identify functional modules. RESULTS The BioNet package provides an extensive framework for integrated network analysis in R. This includes the statistics for the integration of transcriptomic and functional data with biological networks, the scoring of nodes as well as methods for network search and visualization. AVAILABILITY The BioNet package and a tutorial are available from http://bionet.bioapps.biozentrum.uni-wuerzburg.de.

Mutational dynamics between primary and relapse neuroblastomas

Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.

Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

BackgroundTardigrades represent an animal phylum with extraordinary resistance to environmental stress.ResultsTo 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.ConclusionDifferent protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.

Effects of Silver Nitrate and Silver Nanoparticles on a Planktonic Community: General Trends after Short-Term Exposure

Among metal pollutants silver ions are one of the most toxic forms, and have thus been assigned to the highest toxicity class. Its toxicity to a wide range of microorganisms combined with its low toxicity to humans lead to the development of a wealth of silver-based products in many bactericidal applications accounting to more than 1000 nano-technology-based consumer products. Accordingly, silver is a widely distributed metal in the environment originating from its different forms of application as metal, salt and nanoparticle. A realistic assessment of silver nanoparticle toxicity in natural waters is, however, problematic and needs to be linked to experimental approaches. Here we apply metatranscriptome sequencing allowing for elucidating reactions of whole communities present in a water sample to stressors. We compared the toxicity of ionic silver and ligand-free silver nanoparticles by short term exposure on a natural community of aquatic microorganisms. We analyzed the effects of the treatments on metabolic pathways and species composition on the eukaryote metatranscriptome level in order to describe immediate molecular responses of organisms using a community approach. We found significant differences between the samples treated with 5 µg/L AgNO3 compared to the controls, but no significant differences in the samples treated with AgNP compared to the control samples. Statistical analysis yielded 126 genes (KO-IDs) with significant differential expression with a false discovery rate (FDR) <0.05 between the control (KO) and AgNO3 (NO3) groups. A KEGG pathway enrichment analysis showed significant results with a FDR below 0.05 for pathways related to photosynthesis. Our study therefore supports the view that ionic silver rather than silver nanoparticles are responsible for silver toxicity. Nevertheless, our results highlight the strength of metatranscriptome approaches for assessing metal toxicity on aquatic communities.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1−/−‐ and Tlr8−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

Transcriptome Analysis in Tardigrade Species Reveals Specific Molecular Pathways for Stress Adaptations

Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant.

Robustness and accuracy of functional modules in integrated network analysis

MOTIVATION High-throughput molecular data provide a wealth of information that can be integrated into network analysis. Several approaches exist that identify functional modules in the context of integrated biological networks. The objective of this study is 2-fold: first, to assess the accuracy and variability of identified modules and second, to develop an algorithm for deriving highly robust and accurate solutions. RESULTS In a comparative simulation study accuracy and robustness of the proposed and established methodologies are validated, considering various sources of variation in the data. To assess this variation, we propose a jackknife resampling procedure resulting in an ensemble of optimal modules. A consensus approach summarizes the ensemble into one final module containing maximally robust nodes and edges. The resulting consensus module identifies and visualizes robust and variable regions by assigning support values to nodes and edges. Finally, the proposed approach is exemplified on two large gene expression studies: diffuse large B-cell lymphoma and acute lymphoblastic leukemia.

Comprehensive transcriptome analysis provides new insights into nutritional strategies and phylogenetic relationships of chrysophytes

Background Chrysophytes are protist model species in ecology and ecophysiology and important grazers of bacteria-sized microorganisms and primary producers. However, they have not yet been investigated in detail at the molecular level, and no genomic and only little transcriptomic information is available. Chrysophytes exhibit different trophic modes: while phototrophic chrysophytes perform only photosynthesis, mixotrophs can gain carbon from bacterial food as well as from photosynthesis, and heterotrophs solely feed on bacteria-sized microorganisms. Recent phylogenies and megasystematics demonstrate an immense complexity of eukaryotic diversity with numerous transitions between phototrophic and heterotrophic organisms. The question we aim to answer is how the diverse nutritional strategies, accompanied or brought about by a reduction of the plasmid and size reduction in heterotrophic strains, affect physiology and molecular processes. Results We sequenced the mRNA of 18 chrysophyte strains on the Illumina HiSeq platform and analysed the transcriptomes to determine relations between the trophic mode (mixotrophic vs. heterotrophic) and gene expression. We observed an enrichment of genes for photosynthesis, porphyrin and chlorophyll metabolism for phototrophic and mixotrophic strains that can perform photosynthesis. Genes involved in nutrient absorption, environmental information processing and various transporters (e.g., monosaccharide, peptide, lipid transporters) were present or highly expressed only in heterotrophic strains that have to sense, digest and absorb bacterial food. We furthermore present a transcriptome-based alignment-free phylogeny construction approach using transcripts assembled from short reads to determine the evolutionary relationships between the strains and the possible influence of nutritional strategies on the reconstructed phylogeny. We discuss the resulting phylogenies in comparison to those from established approaches based on ribosomal RNA and orthologous genes. Finally, we make functionally annotated reference transcriptomes of each strain available to the community, significantly enhancing publicly available data on Chrysophyceae. Conclusions Our study is the first comprehensive transcriptomic characterisation of a diverse set of Chrysophyceaen strains. In addition, we showcase the possibility of inferring phylogenies from assembled transcriptomes using an alignment-free approach. The raw and functionally annotated data we provide will prove beneficial for further examination of the diversity within this taxon. Our molecular characterisation of different trophic modes presents a first such example.

Integrated pathway modules using time-course metabolic profiles and EST data from Milnesium tardigradum

BackgroundTardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress.ResultsIn this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurable metabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration.ConclusionsThe functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.

Trade‐off between taxon diversity and functional diversity in European lake ecosystems

Inferring ecosystem functioning and ecosystem services through inspections of the species inventory is a major aspect of ecological field studies. Ecosystem functions are often stable despite considerable species turnover. Using metatranscriptome analyses, we analyse a thus‐far unparalleled freshwater data set which comprises 21 mainland European freshwater lakes from the Sierra Nevada (Spain) to the Carpathian Mountains (Romania) and from northern Germany to the Apennines (Italy) and covers an altitudinal range from 38 m above sea level (a.s.l) to 3110 m a.s.l. The dominant taxa were Chlorophyta and streptophytic algae, Ciliophora, Bacillariophyta and Chrysophyta. Metatranscriptomics provided insights into differences in community composition and into functional diversity via the relative share of taxa to the overall read abundance of distinct functional genes on the ecosystem level. The dominant metabolic pathways in terms of the fraction of expressed sequences in the cDNA libraries were affiliated with primary metabolism, specifically oxidative phosphorylation, photosynthesis and the TCA cycle. Our analyses indicate that community composition is a good first proxy for the analysis of ecosystem functions. However, differential gene regulation modifies the relative importance of taxa in distinct pathways. Whereas taxon composition varies considerably between lakes, the relative importance of distinct metabolic pathways is much more stable, indicating that ecosystem functioning is buffered against shifts in community composition through a functional redundancy of taxa.