High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

INTRODUCTION: MicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. OBJECTIVE: The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. METHODS: Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. RESULTS: Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss. CONCLUSIONS: This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.

SNP Discovery in European Anchovy (Engraulis encrasicolus, L) by High-Throughput Transcriptome and Genome Sequencing

12 p.

Contributions to High-Throughput Computing Based on the Peer-to-Peer Paradigm

XII, 116 p.

Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex

9 p. : il.

Wide Screening of Phage-Displayed Libraries Identifies Immune Targets in Planta

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 26107 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Sensing parasites: proteomic and advanced bio-detection alternatives

[EN] Parasitic diseases have a great impact in human and animal health. The gold standard for the diagnosis of the majority of parasitic infections is still conventional microscopy, which presents important limitations in terms of sensitivity and specificity and commonly requires highly trained technicians. More accurate molecular-based diagnostic tools are needed for the implementation of early detection, effective treatments and massive screenings with high-throughput capacities. In this respect, sensitive and affordable devices could greatly impact on sustainable control programmes which exist against parasitic diseases, especially in low income settings. Proteomics and nanotechnology approaches are valuable tools for sensing pathogens and host alteration signatures within microfluidic detection platforms. These new devices might provide novel solutions to fight parasitic diseases. Newly described specific parasite derived products with immune-modulatory properties have been postulated as the best candidates for the early and accurate detection of parasitic infections as well as for the blockage of parasite development. This review provides the most recent methodological and technological advances with great potential for biosensing parasites in their hosts, showing the newest opportunities offered by modern “-omics” and platforms for parasite detection and control.

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.