Núria Canela

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.

Proteomic Profile of Unstable Atheroma Plaque: Increased Neutrophil Defensin 1, Clusterin, and Apolipoprotein E Levels in Carotid Secretome

Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

Supramolecular confinement of polymeric electron transfer mediator on gold surface for picomolar detection of DNA

In this work, we describe a novel route for the immobilisation of an electron transfer mediator on electrode surfaces based on the interfacial complexation of a bifunctionalised carboxymethylcellulose (CMC) polymer backbone bearing ferrocene units and a DNA probe on a cyclodextrin-functionalised surface. The interfacial self-assembly has been studied using surface plasmon resonance and electrochemical techniques and the applicability of the modified surface for the construction of an amperometric genosensor was explored for the detection of a celiac disease associated allele. The supramolecular strategy simplifies the operation of the biosensor, only requiring the addition of an enzyme substrate, and the proximity of the mediator to the electrode surface greatly improves the detection limits attained (10 pM) with respect to a similar supramolecular system based on electrochemically inactive adamantane/CD inclusion complexes (80 pM) and requiring addition of the mediator in solution. In addition, the use of the hydrophilic CMC backbone contributes to the elimination of non-specific interactions and to an optimal spacing of the immobilised DNA probes.

Targeted metabolomic approach in men with carotid plaque

Background The aim of the study was to analyse the presence of several metabolites related to atherosclerosis in the plasma of patients with unstable carotid plaque and in the plasma of healthy subjects. Materials and methods We included 20 patients who had undergone carotid endarterectomy and 20 healthy subjects as a control group. All the subjects recruited were male. We used a metabolomic approach with liquid chromatography coupled to mass spectrometry to evaluate plasma metabolite levels in the metabolic pathway involved in the progression of atherosclerotic plaque. Results We observed that circulating levels of 20-HETE were significantly higher in patients with atheroma plaque than in healthy subjects (p = 0.018). No differences were found with regard to the other metabolites analysed. We also conducted a random forest analysis and found that 20-HETE was the main differentiator in the list of selected metabolites. In addition, plasma levels of 20-HETE correlated positively with body mass index (r = 0.427, p = 0.007) and diastolic blood pressure (r = 0.365, p = 0.028). Conclusion This study confirms that of all the molecules studied only 20-HETE is related to carotid plaque. Further studies are needed to compare patients with stable carotid plaque vs. patients with unstable carotid plaque in order to confirm that 20-HETE could be a potential factor related to carotid plaque.

Monitoring and evaluation of the interaction between deoxynivalenol and gut microbiota in Wistar rats by mass spectrometry-based metabolomics and next-generation sequencing

Published evidence has demonstrated the several toxic characteristics of mycotoxins and their considerable risk to human and animal health. One of the most common uncertainties regards whether if very low concentrations of the mycotoxin deoxynivalenol (DON), easily consumed within the Mediterranean Diet, can cause metabolic alterations; some of them produced by the interaction between DON and gut microbiota. Accordingly, faecal samples were collected from Wistar rats that had consumed the mycotoxin DON at low levels (60 and 120 μg kg-1 body weight of DON per day), and were analysed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry detection, in order to monitor the mycotoxin DON and its metabolite de-epoxy deoxynivalenol (DOM-1). The obtained results showed an evolution in DON excretion and the metabolite DOM-1 which has less toxic properties, over the course of the days of the study. To elucidate whether intestinal microbiota had a role in the observed detoxification process, the changes in microbial gut biodiversity were explored through 16s rRNA high throughput sequencing. No main changes were detected but significant increase in Coprococcus genus relative abundance was found. Further studies are needed to confirm if intestinal microbiota composition and function are affected by low mycotoxin concentrations.

Multi-omics approach to elucidate the gut microbiota activity: Metaproteomics and metagenomics connection

Over the last few years, the application of high‐throughput meta‐omics methods has provided great progress in improving the knowledge of the gut ecosystem and linking its biodiversity to host health conditions, offering complementary support to classical microbiology. Gut microbiota plays a crucial role in relevant diseases such as obesity or cardiovascular disease (CVD), and its regulation is closely influenced by several factors, such as dietary composition. In fact, polyphenol‐rich diets are the most palatable treatment to prevent hypertension associated with CVD, although the polyphenol–microbiota interactions have not been completely elucidated. For this reason, the aim of this study was to evaluate microbiota effect in obese rats supplemented by hesperidin, after being fed with cafeteria or standard diet, using a multi meta‐omics approaches combining strategy of metagenomics and metaproteomics analysis. We reported that cafeteria diet induces obesity, resulting in changes in the microbiota composition, which are related to functional alterations at proteome level. In addition, hesperidin supplementation alters microbiota diversity and also proteins involved in important metabolic pathways. Overall, going deeper into strategies to integrate omics sciences is necessary to understand the complex relationships between the host, gut microbiota, and diet.