An EST-based analysis identifies new genes and reveals distinctive gene expression features of Coffea arabica and Coffea canephora

Background: Coffee is one of the world’s most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency. Results: Assembling the expressed sequence tags (ESTs) of C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories. Conclusion: We present the first comprehensive genome-wide transcript profile study of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/ coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance

CRISPLD1: a novel conserved target in the transition to human heart failure

Heart failure is a major health problem worldwide with a significant morbidity and mortality rate. Although studied extensively in animal models, data from patients at the compensated disease stage are lacking. We sampled myocardium biopsies from aortic stenosis patients with compensated hypertrophy and moderate heart failure and used transcriptomics to study the transition to failure. Sequencing and comparative analysis of analogous samples of mice with transverse aortic constriction identified 25 candidate genes with similar regulation in response to pressure overload, reflecting highly conserved molecular processes. The gene cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is upregulated in the transition to failure in human and mouse and its function is unknown. Homology to ion channel regulatory toxins suggests a role in Ca(2+) cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca(2+) handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca(2+)-signaling pathways upon CRISPLD1-KO and its upregulation in the transition to failure implicates a contribution to adverse remodeling. These findings provide new pathophysiological data on Ca(2+) regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions

Iis - Integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools

Background: High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. Results: We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. Conclusions: We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. 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A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) [558272/2009-6]Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB

Transcriptome analysis reveals putative genes involved in the lipid metabolism of chaulmoogra oil biosynthesis in Carpotroche brasiliensis (Raddi) A.Gray, a tropical tree species

Chaulmoogra oil is found in the seeds of Carpotroche brasiliensis (Raddi) Endl. (syn. Mayna brasiliensis Raddi), an oil tree of the Achariaceae family and native to Brazil’s Atlantic Forest biome, which is considered the fifth most important biodiversity hotspot in the world. Its main constituents are cyclopentenic fatty acids. Chaulmoogra oil has economic potential because of its use in the cosmetics industry and as a drug with anti-tumor activity. The mechanisms related to the regulation of oil biosynthesis in C. brasiliensis seeds are not fully understood, especially from a tissue-specific perspective. In this study, we applied a de novo transcriptomic approach to investigate the transcripts involved in the lipid pathways of C. brasiliensis and to identify genes involved in lipid biosynthesis. Comparative analysis of gene orthology, expression analysis and visualization of metabolic lipid networks were performed, using data obtained from high-throughput sequencing (RNAseq) of 24 libraries of vegetative and reproductive tissues of C. brasiliensis. Approximately 10.4 million paired-end reads (Phred (Q) > 20) were generated and re-assembled into 107,744 unigenes, with an average length of 340 base pairs (bp). The analysis of transcripts from different tissues identified 1131 proteins involved in lipid metabolism and transport and 13 pathways involved in lipid biosynthesis, degradation, transport, lipid bodies, and lipid constituents of membranes. This is the first transcriptome study of C. brasiliensis, providing basic information for biotechnological applications of great use for the species, which will help understand chaulmoogra oil biosynthesis

New Insights into White-Light Flare Emission from Radiative-Hydrodynamic Modeling of a Chromospheric Condensation

(abridged) The heating mechanism at high densities during M dwarf flares is poorly understood. Spectra of M dwarf flares in the optical and near-ultraviolet wavelength regimes have revealed three continuum components during the impulsive phase: 1) an energetically dominant blackbody component with a color temperature of T $\sim$ 10,000 K in the blue-optical, 2) a smaller amount of Balmer continuum emission in the near-ultraviolet at lambda $<$ 3646 Angstroms and 3) an apparent pseudo-continuum of blended high-order Balmer lines. These properties are not reproduced by models that employ a typical "solar-type" flare heating level in nonthermal electrons, and therefore our understanding of these spectra is limited to a phenomenological interpretation. We present a new 1D radiative-hydrodynamic model of an M dwarf flare from precipitating nonthermal electrons with a large energy flux of $10^{13}$ erg cm$^{-2}$ s$^{-1}$. The simulation produces bright continuum emission from a dense, hot chromospheric condensation. For the first time, the observed color temperature and Balmer jump ratio are produced self-consistently in a radiative-hydrodynamic flare model. We find that a T $\sim$ 10,000 K blackbody-like continuum component and a small Balmer jump ratio result from optically thick Balmer and Paschen recombination radiation, and thus the properties of the flux spectrum are caused by blue light escaping over a larger physical depth range compared to red and near-ultraviolet light. To model the near-ultraviolet pseudo-continuum previously attributed to overlapping Balmer lines, we include the extra Balmer continuum opacity from Landau-Zener transitions that result from merged, high order energy levels of hydrogen in a dense, partially ionized atmosphere. This reveals a new diagnostic of ambient charge density in the densest regions of the atmosphere that are heated during dMe and solar flares.Comment: 50 pages, 2 tables, 13 figures. Accepted for publication in the Solar Physics Topical Issue, "Solar and Stellar Flares". Version 2 (June 22, 2015): updated to include comments by Guest Editor. The final publication is available at Springer via http://dx.doi.org/10.1007/s11207-015-0708-

Comparative analysis of the secretome and interactome of Trypanosoma cruzi and Trypanosoma rangeli reveals species specific immune response modulating proteins

Chagas disease, a zoonosis caused by the flagellate protozoan Trypanosoma cruzi, is a chronic and systemic parasitic infection that affects ~5–7 million people worldwide, mainly in Latin America. Chagas disease is an emerging public health problem due to the lack of vaccines and effective treatments. According to recent studies, several T. cruzi secreted proteins interact with the human host during cell invasion. Moreover, some comparative studies with T. rangeli, which is non-pathogenic in humans, have been performed to identify proteins directly involved in the pathogenesis of the disease. In this study, we present an integrated analysis of canonical putative secreted proteins (PSPs) from both species. Additionally, we propose an interactome with human host and gene family clusters, and a phylogenetic inference of a selected protein. In total, we identified 322 exclusively PSPs in T. cruzi and 202 in T. rangeli. Among the PSPs identified in T. cruzi, we found several trans-sialidases, mucins, MASPs, proteins with phospholipase 2 domains (PLA2-like), and proteins with Hsp70 domains (Hsp70-like) which have been previously characterized and demonstrated to be related to T. cruzi virulence. PSPs found in T. rangeli were related to protozoan metabolism, specifically carboxylases and phosphatases. Furthermore, we also identified PSPs that may interact with the human immune system, including heat shock and MASP proteins, but in a lower number compared to T. cruzi. Interestingly, we describe a hypothetical hybrid interactome of PSPs which reveals that T. cruzi secreted molecules may be down-regulating IL-17 whilst T. rangeli may enhance the production of IL-15. These results will pave the way for a better understanding of the pathophysiology of Chagas disease and may ultimately lead to the identification of molecular targets, such as key PSPs, that could be used to minimize the health outcomes of Chagas disease by modulating the immune response triggered by T. cruzi infection

The Genome Sequence Of Leishmania (leishmania) Amazonensis: Functional Annotation And Extended Analysis Of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. 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Dissecting herpes simplex virus 1-induced host shutoff at the RNA level

Herpes simplex virus 1 (HSV-1) induces a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and Δvhs infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. In parallel, host transcriptional activity dropped to 10-20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infection. Both induced strong transcriptional up-regulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional down-regulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8h of lytic HSV-1 infection. IMPORTANCE: The HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity as well as virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes