Rosario Carmona

A microRNA Signature Associated with Early Recurrence in Breast Cancer

Recurrent breast cancer occurring after the initial treatment is associated with poor outcome. A bimodal relapse pattern after surgery for primary tumor has been described with peaks of early and late recurrence occurring at about 2 and 5 years, respectively. Although several clinical and pathological features have been used to discriminate between low- and high-risk patients, the identification of molecular biomarkers with prognostic value remains an unmet need in the current management of breast cancer. Using microarray-based technology, we have performed a microRNA expression analysis in 71 primary breast tumors from patients that either remained disease-free at 5 years post-surgery (group A) or developed early (group B) or late (group C) recurrence. Unsupervised hierarchical clustering of microRNA expression data segregated tumors in two groups, mainly corresponding to patients with early recurrence and those with no recurrence. Microarray data analysis and RT-qPCR validation led to the identification of a set of 5 microRNAs (the 5-miRNA signature) differentially expressed between these two groups: miR-149, miR-10a, miR-20b, miR-30a-3p and miR-342-5p. All five microRNAs were down-regulated in tumors from patients with early recurrence. We show here that the 5-miRNA signature defines a high-risk group of patients with shorter relapse-free survival and has predictive value to discriminate non-relapsing versus early-relapsing patients (AUC = 0.993, p-value<0.05). Network analysis based on miRNA-target interactions curated by public databases suggests that down-regulation of the 5-miRNA signature in the subset of early-relapsing tumors would result in an overall increased proliferative and angiogenic capacity. In summary, we have identified a set of recurrence-related microRNAs with potential prognostic value to identify patients who will likely develop metastasis early after primary breast surgery.

ReprOlive: a database with linked data for the olive tree (Olea europaea L.) reproductive transcriptome.

Plant reproductive transcriptomes have been analyzed in different species due to the agronomical and biotechnological importance of plant reproduction. Here we presented an olive tree reproductive transcriptome database with samples from pollen and pistil at different developmental stages, and leaf and root as control vegetative tissues http://reprolive.eez.csic.es). It was developed from 2,077,309 raw reads to 1,549 Sanger sequences. Using a pre-defined workflow based on open-source tools, sequences were pre-processed, assembled, mapped, and annotated with expression data, descriptions, GO terms, InterPro signatures, EC numbers, KEGG pathways, ORFs, and SSRs. Tentative transcripts (TTs) were also annotated with the corresponding orthologs in Arabidopsis thaliana from TAIR and RefSeq databases to enable Linked Data integration. It results in a reproductive transcriptome comprising 72,846 contigs with average length of 686 bp, of which 63,965 (87.8%) included at least one functional annotation, and 55,356 (75.9%) had an ortholog. A minimum of 23,568 different TTs was identified and 5,835 of them contain a complete ORF. The representative reproductive transcriptome can be reduced to 28,972 TTs for further gene expression studies. Partial transcriptomes from pollen, pistil, and vegetative tissues as control were also constructed. ReprOlive provides free access and download capability to these results. Retrieval mechanisms for sequences and transcript annotations are provided. Graphical localization of annotated enzymes into KEGG pathways is also possible. Finally, ReprOlive has included a semantic conceptualisation by means of a Resource Description Framework (RDF) allowing a Linked Data search for extracting the most updated information related to enzymes, interactions, allergens, structures, and reactive oxygen species.

Identification of Distinctive Variants of the Olive Pollen Allergen Ole e 5 (Cu,Zn Superoxide Dismutase) throughout the Analysis of the Olive Pollen Transcriptome

Ole e 5 is an olive pollen allergen displaying high identity with Cu,Zn superoxide dismutases. Previous studies characterized biochemical variability in this allergen, which may be of relevance for allergy diagnosis and therapy. The generation of an olive pollen transcriptome allowed us to identify eight Ole e 5 sequences, one of them including a 24 nt deletion. Further in silico analysis permitted designing primers for PCR amplification and cloning from both cDNA and gDNA. A large number of sequences were retrieved, which experimentally validated the predictive NGS sequences, including the deleted enzyme. The PCR-obtained sequences were used for further scrutiny, including sequence aligment and phylogenetic analysis. Two model sequences (a complete sequence and a deleted one) were used to perform 3-D modeling and a prediction of the T- and B-cell epitopes. These predictions interestingly foreseed relevant differences in the antigenicity/allergenicity of both molecules. Clinical relevance of differences is discussed.

Identification and in silico Analysis of Glutathione Reductase Transcripts Expressed in Olive (Olea europaea L.) Pollen and Pistil

Glutathione (GSH) protects proteins against oxidation of their thiol-containing groups, by alternatively becoming the subject of oxidation, forming glutathione disulfide (GSSG). Appropriate GSH:GSSG levels are maintained by glutathione reductase (GR), a homodimeric flavoprotein which uses NADPH to reduce one GSSG molecule to two of GSH. This enzyme has been characterized in several species, and described as highly conserved, with two isoforms only. Heterogeneity and discticntiveness of plant reproductive tissues led us to investigate the presence of GR sequences. A de novo assembled and annotated olive reproductive transcriptome was subjected to screening, which allowed us to identify at least 11 GR homologues (1 pollen-specific and 10 from pistils). Primers were designed, and full-length sequences were obtained through PCR. In silico analysis, including phylogeny, 3-D modeling of N-terminus, and prediction of cellular localization and post-translational modifications was carried out to shed light into the involvement of olive pollen-intrinsic GR in reproductive development.

Identification and in silico Analysis of NADPH Oxidase Homologues Involved in Allergy from an Olive Pollen Transcriptome

Reactive oxygen species generated by pollen NADPH oxidases are present in numerous allergenic pollen species. The superoxide generated by this enzyme has been suggested as a key actor in the induction of allergic inflammation. However, this enzyme has been characterized in Arabidopsis thaliana pollen only, where two pollen-specific genes (RbohH and RbohJ) have been described. The olive (Olea europaea L.) pollen is an important source of allergy in Mediterranean countries. We have assembled and annotated an olive pollen transcriptome, which allowed us to determine the presence of at least two pollen-specific NADPH oxidase homologues. Primers were designed to distinguish between the two homologues, and full-length sequences were obtained through a PCR strategy. Complete in silico analysis of such sequences, including phylogeny, 3-D modeling of the N-terminus, and prediction of cellular localization and post-translational modifications was carried out with the purpose of shed light into the involvement of olive pollen-intrinsic NADPH oxidases in triggering allergy symptoms.