Publications

Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer ' s disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrP C ) during disease progression. That is why, by means of cross studies of miRNAs up -regulated in AD with in silico identification of potential miRNAs-binding to 3 ' UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3 ' UTR- PRNP , and second, we analyzed the levels of PrP C expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non -AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3 ' UTR in vitro and promotes downregulation of PrP C . Moreover, miR-519a-3p was found to be up -regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.

JTD Keywords: 3' untranslated regions, Activation, Aged, Aged, 80 and over, Alzheimer disease, Alzheimer's disease, Biomarke, Biomarker, Biomarkers, Brain, Cellular prion protein, Developmental expression, Female, Gen, Humans, Male, Micrornas, Mirn519 microrna, human, Plasma, Prion proteins, Prnp protein, human, Prpc, Prpc proteins, Tau-aggregation

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3?UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3?UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3?UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

JTD Keywords: aggregative adherence, arginine metabolism, biofilm formation, escherichia-coli, gene-expression, messenger-rna, operon, persistent diarrhea, untranslated region, Aggr protein, e coli, Escherichia coli, Escherichia coli proteins, Fimbria-i expression, Trans-activators, Virulence

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

JTD Keywords: expression, in-vitro, mouse model, muscle, mutations, phenotype, quantification, sarcolemma, therapy, 3' untranslated regions, Cells, cultured, Crispr-cas systems, Cytoskeletal proteins, Drug discovery, Dystroglycans, Dystrophin, Gene editing, Hek293 cells, Humans, Muscular dystrophy, duchenne, Myf5 protein, human, Myh3 polypeptide, human, Myoblasts, Myogenic regulatory factor 5, Primary cell culture, Sarcoglycans, Utrophin, Utrophin up-regulation