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Zubiri I.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Vivanco F.,IIS Fundacion Jimenez Diaz | Vivanco F.,Complutense University of Madrid | Gloria A.-L.,IIS Fundacion Jimenez Diaz
Methods in Molecular Biology | Year: 2013

Urinary exosomes are membranous vesicles 40-100 nm in size containing proteins that are characteristic of every renal tubule epithelial cell type. In this chapter, we describe a methodology to isolate and analyze urinary exosomes proteome by 2-DE and LC-MS/MS, in the search for biomarkers of vascular and associated kidney diseases. We describe an isolation methodology by serial (ultra)centrifugation steps compatible with 2-DE and LC-MS/MS analysis. Exosome purity is con fi rmed by electron microscopy and Western blot. © 2013 Springer Science+Business Media New York.


Tunon J.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Tunon J.,Autonomous University of Madrid | Martin-Ventura J.L.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Martin-Ventura J.L.,Autonomous University of Madrid | And 7 more authors.
Journal of the American College of Cardiology | Year: 2010

Extensive research has focused on the identification of novel plasma biomarkers to improve our ability to predict cardiovascular events in atherothrombosis. However, classical techniques can only assess a limited number of proteins at a time. Given that plasma contains more than 900,000 proteins, this approach will be extremely time-consuming. Novel proteomic approaches make it possible to compare the expression of hundreds of proteins in several samples in a single experiment. The classical approach consists of separation of proteins on a 2-dimensional gel followed by protein identification with mass spectrometry, although new complementary gel-free techniques are emerging. We can thus compare protein expression in an atherosclerotic plaque with that in a normal artery or study plasma proteins in patients with atherothrombosis as compared with healthy subjects. For such approaches, it is not necessary to study the published data to select potential biomarkers. However, because the number of patients that can be studied with most of these techniques is limited, what is really important is the design of the studies, assessing carefully what kind of patients should be included to obtain valid conclusions. Clinicians should thus play a key role in this design along with the basic scientist. In this article, we review several proteomic strategies carried out by our group and others, and we make a call for collaboration between clinicians and experts in proteomics. This collaboration could greatly increase the likelihood of identifying new prognostic biomarker panels in atherothrombosis and other cardiovascular disorders. © 2010 American College of Cardiology Foundation.


Llorente-Folch I.,Autonomous University of Madrid | Llorente-Folch I.,Research Center Biomedica En Red Of Enfermedades Raras Ciberer | Llorente-Folch I.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Rueda C.B.,Autonomous University of Madrid | And 11 more authors.
Journal of Physiology | Year: 2015

Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. Although restoring ionic gradients is energetically costly, a rise in intracellular Ca2+ acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca2+ stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca2+-regulated mitochondrial carriers, whereas an increase in matrix Ca2+ stimulates the citric acid cycle and ATP synthase. The aspartate-glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate-aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca2+ and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K+-depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 knockout neurons reflect impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca2+ influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca2+]. Although the physiological importance of the MCU complex in work-induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca2+ signalling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCU complex, are associated with neuromuscular disease. In patient-derived MICU1 deficient fibroblasts, resting matrix Ca2+ is increased and mitochondria fragmented. Thus, the fine tuning of Ca2+ signals plays a key role in shaping mitochondrial bioenergetics. © 2015 The Physiological Society.


Ucero A.C.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Berzal S.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Ocana-Salceda C.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Sancho M.,Research Center Principe Felipe | And 8 more authors.
PLoS ONE | Year: 2013

The polyglutamic acid/peptoid 1 (QM56) nanoconjugate inhibits apoptosis by interfering with Apaf-1 binding to procaspase-9. We now describe anti-inflammatory properties of QM56 in mouse kidney and renal cell models. In cultured murine tubular cells, QM56 inhibited the inflammatory response to Tweak, a non-apoptotic stimulus. Tweak induced MCP-1 and Rantes synthesis through JAK2 kinase and NF-κB activation. Similar to JAK2 kinase inhibitors, QM56 inhibited Tweak-induced NF-κB transcriptional activity and chemokine expression, despite failing to inhibit NF-κB-p65 nuclear translocation and NF-κB DNA binding. QM56 prevented JAK2 activation and NF-κB-p65(Ser536) phosphorylation. The anti-inflammatory effect and JAK2 inhibition by QM56 were observed in Apaf-1-/- cells. In murine acute kidney injury, QM56 decreased tubular cell apoptosis and kidney inflammation as measured by down-modulations of MCP-1 and Rantes mRNA expression, immune cell infiltration and activation of the JAK2-dependent inflammatory pathway. In conclusion, QM56 has an anti-inflammatory activity which is independent from its role as inhibitor of Apaf-1 and apoptosis and may have potential therapeutic relevance. © 2013 Ucero et al.


PubMed | Autonomous University of Madrid and Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD
Type: | Journal: Scientific reports | Year: 2016

The introduction of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus greatly reduced the rate of allograft rejection, although their chronic use is marred by a range of side effects, among them vascular toxicity. In transplant patients, it is proved that innate immunity promotes vascular injury triggered by ischemia-reperfusion damage, atherosclerosis and hypertension. We hypothesized that activation of the innate immunity and inflammation may contribute to CNI toxicity, therefore we investigated whether TLR4 mediates toxic responses of CNIs in the vasculature. Cyclosporine and tacrolimus increased the production of proinflammatory cytokines and endothelial activation markers in cultured murine endothelial and vascular smooth muscle cells as well as in ex vivo cultures of murine aortas. CNI-induced proinflammatory events were prevented by pharmacological inhibition of TLR4. Moreover, CNIs were unable to induce inflammation and endothelial activation in aortas from TLR4(-/-) mice. CNI-induced cytokine and adhesion molecules synthesis in endothelial cells occurred even in the absence of calcineurin, although its expression was required for maximal effect through upregulation of TLR4 signaling. CNI-induced TLR4 activity increased O2(-)/ROS production and NF-B-regulated synthesis of proinflammatory factors in cultured as well as aortic endothelial and VSMCs. These data provide new insight into the mechanisms associated with CNI vascular inflammation.


Rio P.,CIEMAT | Rio P.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | Banos R.,CIEMAT | Banos R.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | And 30 more authors.
EMBO Molecular Medicine | Year: 2014

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.


Khateb S.,Hebrew University of Jerusalem | Zelinger L.,Hebrew University of Jerusalem | Mizrahi-Meissonnier L.,Hebrew University of Jerusalem | Ayuso C.,Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD | And 5 more authors.
Journal of Medical Genetics | Year: 2014

Background: Usher syndrome (USH) is a heterogeneous group of inherited retinitis pigmentosa (RP) and sensorineural hearing loss (SNHL) caused by mutations in at least 12 genes. Our aim is to identify additional USH-related genes. Methods: Clinical examination included visual acuity test, funduscopy and electroretinography. Genetic analysis included homozygosity mapping and whole exome sequencing (WES). Results: A combination of homozygosity mapping and WES in a large consanguineous family of Iranian Jewish origin revealed nonsense mutations in two ciliary genes: c.3289C>T (p.Q1097*) in C2orf71 and c.3463C>T (p.R1155*) in centrosome-associated protein CEP250 (C-Nap1). The latter has not been associated with any inherited disease and the c.3463C>T mutation was absent in control chromosomes. Patients who were double homozygotes had SNHL accompanied by earlyonset and severe RP, while patients who were homozygous for the CEP250 mutation and carried a single mutant C2orf71 allele had SNHL with mild retinal degeneration. No ciliary structural abnormalities in the respiratory system were evident by electron microscopy analysis. CEP250 expression analysis of the mutant allele revealed the generation of a truncated protein lacking the NEK2-phosphorylation region. Conclusions: A homozygous nonsense CEP250 mutation, in combination with a heterozygous C2orf71 nonsense mutation, causes an atypical form of USH, characterised by early-onset SNHL and a relatively mild RP. The severe retinal involvement in the double homozygotes indicates an additive effect caused by nonsense mutations in genes encoding ciliary proteins.


PubMed | University of Castilla - La Mancha, Hospital Clinico San Carlos and Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD
Type: Journal Article | Journal: PloS one | Year: 2015

Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of FOXC1 in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic FOXC1 mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable FOXC1 transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability.


PubMed | University of Lausanne, Institute Investigacion Sanitaria Fundacion Jimenez Diaz IIS FJD, Fundacion Jimenez Diaz University Hospital and French Institute of Health and Medical Research
Type: Journal Article | Journal: PloS one | Year: 2016

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

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