VirtualPlant: A Software Platform to Support Systems Biology Research

Data generation is no longer the limiting factor in advancing biological research. In addition, data integration, analysis, and interpretation have become key bottlenecks and challenges that biologists conducting genomic research face daily. To enable biologists to derive testable hypotheses from the increasing amount of genomic data, we have developed the VirtualPlant software platform. VirtualPlant enables scientists to visualize, integrate, and analyze genomic data from a systems biology perspective. VirtualPlant integrates genome-wide data concerning the known and predicted relationships among genes, proteins, and molecules, as well as genome-scale experimental measurements. VirtualPlant also provides visualization techniques that render multivariate information in visual formats that facilitate the extraction of biological concepts. Importantly, VirtualPlant helps biologists who are not trained in computer science to mine lists of genes, microarray experiments, and gene networks to address questions in plant biology, such as: What are the molecular mechanisms by which internal or external perturbations affect processes controlling growth and development? We illustrate the use of VirtualPlant with three case studies, ranging from querying a gene of interest to the identification of gene networks and regulatory hubs that control seed development. Whereas the VirtualPlant software was developed to mine Arabidopsis (Arabidopsis thaliana) genomic data, its data structures, algorithms, and visualization tools are designed in a species-independent way. VirtualPlant is freely available at www.virtualplant.org.Today, experimental biology laboratories usually investigate the molecular mechanisms underlying a physiological or developmental response by identifying the genes involved using a genomic platform, such as microarray (or, soon, deep sequencing) technology. Such a platform might identify genes regulated during a physiological or developmental response. Once the relevant gene sets are identified, biologists next analyze their functional relationships (e.g. whether they belong to the same metabolic pathway) and analyze their properties in the context of known biological pathways (DeRisi et al., 1997). Performing these tasks can be cumbersome because the biologist has to use several different tools to accomplish them. In addition, the difficulty is often increased because the different tools do not read and write the same data formats, forcing the biologist to obtain data conversion software.Aside from the challenge of integrating the vast amount of knowledge accumulated in the literature about the relevant genes, the genomic data available in the public domain have been obtained with a large number of experimental approaches and an even larger number of laboratories. Moreover, the information is stored in numerous databases, and it is encoded in diverse formats and database schemas. Bioinformatics faces a major challenge integrating this large-scale, heterogeneous information into architectures that support biological research. Different approaches that have been employed include hypertext navigation on the World Wide Web, data warehousing, and client-side integration (for example, see Ritter, 1994; Karp, 1996; Siepel et al., 2001; Philippi, 2004; Wilkinson et al., 2005). Once data from distinct database sources are coherently integrated, tools and computer models can be used to enable one to visualize and analyze this biological data from a systems perspective (Ideker et al., 2001). Several environments have been developed to support data integration and modeling (Kahlem and Birney, 2007). Some software allows detailed mathematical representation of cellular processes (e.g. Gepasi [Mendes, 1997] and Virtual Cell [Loew and Schaff, 2001]), while other software permits qualitative representations of cellular components and their interactions (e.g. Cytoscape [Shannon et al., 2003], Osprey [Breitkreutz et al., 2003], and N-Browse [Kao and Gunsalus, 2008]). Generally, quantitative models build detailed mathematical abstractions of specific cellular process. Quantitative models are powerful because they describe a system in detail (Endy and Brent, 2001), but they require a detailed understanding of the system. Unfortunately, this information is available for only a few biological processes. In fact, there are still many gaps in our qualitative understanding of biological systems, even for model organisms. For example, most of the genes in Arabidopsis (Arabidopsis thaliana) have not yet been experimentally characterized. Thus, while quantitative computer models can provide powerful, detailed representations of biological systems, not enough is known about Arabidopsis and other plants to construct such models of them or their major components. Therefore, we have focused on building software that facilitates analysis of the systems and statistical and interaction relationships between their genes and gene products.Today''s most widely available measure of gene function is the level of gene expression provided by a microarray analysis. Many approaches and tools support analysis of expression data. A now classic approach, for example, is to identify genes that are coregulated in their expression patterns across selected experimental conditions (e.g. Eisen et al., 1998). An extensive review of the different software tools that are available for studying gene coexpression is available (Usadel et al., 2009). To identify genes that are differentially expressed between two experimental conditions, statistical methods such as Rank Products can be used (Breitling et al., 2004; Hong et al., 2006). Several tools are available as packages in BioConductor, a project largely composed of tools written in the statistical language R (Gentleman et al., 2004). To determine the biological significance of differentially or coexpressed genes, biologists often evaluate the frequency of occurrence of functional attributes provided by structured functional annotations, such as Gene Ontology (GO; Ashburner et al., 2000). Several software packages to automate this type of analysis now exist (e.g. Onto-Express [Khatri et al., 2002], GoMiner [Zeeberg et al., 2003], GOSurfer [Zhong et al., 2004], and FatiGO [Al-Shahrour et al., 2004]). While advanced data analysis tools for exploiting genomic data are rapidly emerging (for review, see Brady and Provart, 2009), the narrow specialization of most current software tools forces geneticists to employ many tools to analyze the data in a single biological study. This cumbersome and inefficient process greatly hinders biologists following a systems approach of iterative in silico exploration and experimentation.VirtualPlant addresses these problems by integrating selected genomic data and analysis tools into a single Web-accessible software platform. The goal of our work is to help biologists discover new insights by synthesizing multiple data sources. VirtualPlant provides access to a database storing selected information about Arabidopsis and rice (Oryza sativa) experiments, genes, gene products, and their properties. VirtualPlant''s software architecture and data model have been designed and created in a generic, species-independent manner to ease the addition of new organisms and tools in the future. The VirtualPlant database also includes a high-level representation of plant cellular components and interactions that allow users to create molecular networks “on the fly.” These molecular networks provide a framework for analyzing experimental measurements. VirtualPlant also includes novel data visualization and data analysis techniques that allow seamless information exploration across many data sets with the help of a shopping cart in which gene sets from experiments and/or analyses can be stored and then used as inputs to other tools to enable iterative analysis. For concreteness, we present an example of how we have used VirtualPlant to identify gene networks and putative regulatory hubs that control seed development. We have previously demonstrated the use of VirtualPlant and specific tools embodied in the VirtualPlant system to generate hypotheses that were validated experimentally (Wang et al., 2004; Gutiérrez et al., 2007b, 2008; Gifford et al., 2008; Thum et al., 2008).     

Asymmetrical dimethylarginine plasma clearance persists after acute total nephrectomy in rats

Elevated plasma concentrations of symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA) are repeatedly associated with kidney failure. Both ADMA and SDMA can be excreted in urine. We tested whether renal excretion is necessary for acute, short-term maintenance of plasma ADMA and SDMA. Sprague-Dawley rats underwent sham operation, bilateral nephrectomy (NPX), ureteral ligation, or ureteral section under isoflurane anesthesia. Tail-snip blood samples (250 microl) were taken before and at 6- or 12-h intervals for 72 h after operation. Plasma clearance was assessed in intact and NPX rats. High-performance liquid chromatography determined SDMA and ADMA concentrations. Sodium, potassium, creatinine, blood urea nitrogen (BUN), and body weight were also measured. Forty-eight hours after NPX, SDMA increased 25 times (0.23 +/- 0.03 to 5.68 +/- 0.30 microM), whereas ADMA decreased (1.17 +/- 0.08 to 0.73 +/- 0.08 microM) by 38%. Creatinine and BUN increased, paralleling SDMA. Sham-operated animals showed no significant changes. Increased SDMA confirms continuous systemic production of SDMA and its obligatory renal excretion, much like creatinine. In contrast, decreased plasma ADMA suggests that acute total NPX either reduced systemic ADMA formation and/or systemic hydrolysis of ADMA increased 48-h post-NPX. However, plasma clearance of ADMA appeared unchanged 48 h after NPX. We conclude that renal excretory function is needed for SDMA elimination but not needed for acute, short-term ADMA elimination in that systemic hydrolysis is fully capable of clearing plasma ADMA.     

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley Fever vaccine in mice

Background

Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens.

Methods

Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice.

Results

A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8⁺ T cell response.

Conclusions

Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

     

Expressed sequence tag analysis in <Emphasis Type="Italic">Cycas</Emphasis>, the most primitive living seed plant

Background

Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors.

Results

A total of 4,200 expressed sequence tags (ESTs) were created from Cycas rumphii and clustered into 2,458 contigs, of which 1,764 had low-stringency BLAST similarity to other plant genes. Among those cycad contigs with similarity to plant genes, 1,718 cycad 'hits' are to angiosperms, 1,310 match genes in gymnosperms and 734 match lower (non-seed) plants. Forty-six contigs were found that matched only genes in lower plants and gymnosperms. Upon obtaining the complete sequence from the clones of 37/46 contigs, 14 still matched only gymnosperms. Among those cycad contigs common to higher plants, ESTs were discovered that correspond to those involved in development and signaling in present-day flowering plants. We purified a cycad EST for a glutamate receptor (GLR)-like gene, as well as ESTs potentially involved in the synthesis of the GLR agonist BMAA.

Conclusions

Analysis of cycad ESTs has uncovered conserved and potentially novel genes. Furthermore, the presence of a glutamate receptor agonist, as well as a glutamate receptor-like gene in cycads, supports the hypothesis that such neuroactive plant products are not merely herbivore deterrents but may also serve a role in plant signaling.

     

Identification of mesoderm development (mesd) candidate genes by comparative mapping and genome sequence analysis

The proximal albino deletions identify several functional regions on mouse Chromosome 7 critical for differentiation of mesoderm (mesd), development of the hypothalamus neuroendocrine lineage (nelg), and function of the liver (hsdr1). Using comparative mapping and genomic sequence analysis, we have identified four novel genes and Il16 in the mesd deletion interval. Two of the novel genes, mesdc1 and mesdc2, are located within the mesd critical region defined by BAC transgenic rescue. We have investigated the fetal role of genes located outside the mesd critical region using BAC transgenic complementation of the mesd early embryonic lethality. Using human radiation hybrid mapping and BAC contig construction, we have identified a conserved region of human chromosome 15 homologous to the mesd, nelg, and hsdr1 functional regions. Three human diseases cosegregate with microsatellite markers used in construction of the human BAC/YAC physical map, including autosomal dominant nocturnal frontal lobe epilepsy (ENFL2; also known as ADNFLE), a syndrome of mental retardation, spasticity, and tapetoretinal degeneration (MRST); and a pyogenic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA).     

Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.