Proyectos

Endothelial injury, inflammatory infiltrate and fibrosis are the predominant lesions in the testis of bulls with besnoitiosis that may result in sterility. Moreover, fibroblasts, which are key players in fibrosis, are parasite target cells in a Besnoitia besnoiti chronic infection. This study aimed to decipher the molecular basis that underlies a drift toward fibrosis during the disease progression. Transcriptomic analysis was developed at two times post-infection (p.i.), representative of invasion (12 h p.i.) and intracellular proliferation (32 h p.i.), in primary bovine aorta fibroblasts infected with B. besnoiti tachyzoites. Once the enriched host pathways were identified, we studied the expression of selected differentially expressed genes (DEGs) in the scrotal skin of sterile infected bulls. Functional enrichment analyses of DEGs revealed shared hallmarks of cancer and early fibrosis. Biomarkers of inflammation, angiogenesis, cancer, and MAPK signaling stood out at 12 h p.i. At 32 h p.i., again MAPK and cancer pathways were enriched together with the PI3K–AKT pathway related to cell proliferation. Some DEGs were also regulated in the skin samples of naturally infected bulls (PLAUR, TGFβ1, FOSB). We have identified potential biomarkers and host pathways regulated during fibrosis that may hold prognostic significance and could emerge as potential therapeutic targets.

María Fernández-Álvarez, Pilar Horcajo , Alejandro Jiménez-Meléndez, Pablo Angulo Lara, Ana Huertas-López, Francisco Huertas-López, Ignacio Ferre, Luis Miguel Ortega-Mora and Gema Álvarez-García

En la era de la medicina personalizada, las tecnologías de secuenciación y las herramientas ómicas han revoucionado la comprensión de las enfermedades, destacando la importancia de la microbiota intestinal en la salud y la enfermedad. Este estudio se centra en la relación entre la microbiota intestinal y dos enfermedades cutáneas inflamatorias: dermatitis atópica y alopecia areata. El objetivo es profundizar en estas conexiones a través de estudios bioinformáticos que permitan desarrollar un modelo de inteligencia artificial para su clasificación basada en icroorganismos específicos.

Sánchez Pellicer P, Núñez Delegido E, Navarro Moratalla L, Agüera Santos JG, Serrano López EM, Huertas López F, Navarro López V

A lo largo del tracto reproductor femenino, el espermatozoide sufre una serie de cambios necesarios para que la fecundación tenga lugar, conocidos como capacitación. Uno de ellos es la hiperactivación, caracterizada por un cambio en el movimiento espermático necesario para que el espermatozoide pueda atravesar la zona pelúcida del óvulo. La hiperactivación se puede determinar de un modo no invasivo mediante el sistema automático CASA (Computer Assisted Sperm Analysis). El patrón de motilidad de los espermatozoides es heterogéneo y complejo, por ello el uso de modelos de inteligencia artificial (IA) resultan útiles a la hora de identificar parámetros relacionados con la hiperactivación, permitiendo el desarrollo de modelos computacionales que ayuden a la predicción de la calidad seminal y su potencial aplicación en la predicción del éxito en la inseminación artificial.

Paula Cots Rodríguez, T Casas Pina, EM Serrano López, M Avilés González, F Huertas López, J Gálvez Pradillo, J Álvarez Castillo, M Avilés

Controlled ovarian stimulation is a necessary step in some assisted reproductive procedures allowing a higher collection of female gametes. However, consequences of this stimulation for the gamete or the offspring have been shown in several mammals. Most studies used comparisons between oocytes from different donors, which may contribute to different responses. In this work, we use the bovine model in which each animal serves as its own control. DNA methylation profiles were obtained by single-cell whole-genome bisulfite sequencing of oocytes from pre-ovulatory unstimulated follicles compared to oocytes from stimulated follicles. Results show that the global percentage of methylation was similar between groups, but the percentage of methylation was lower for non-stimulated oocytes in the imprinted genes APEG3, MEG3, and MEG9 and higher in TSSC4 when compared to stimulated oocytes. Differences were also found in CGI of imprinted genes: higher methylation was found among non-stimulated oocytes in MEST (PEG1), IGF2R, GNAS (SCG6), KvDMR1 ICR UMD, and IGF2. In another region around IGF2, the methylation percentage was lower for non-stimulated oocytes when compared to stimulated oocytes. Data drawn from this study might help to understand the molecular reasons for the appearance of certain syndromes in assisted reproductive technologies-derived offspring.

Jordana S. Lopes, Elena Ivanova, Salvador Ruiz, Simon Andrews, Gavin Kelsey, and Pilar Coy.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.

Emilio M Serrano-López, Teresa Coronado-Parra, Consuelo Marín-Vicente, Zoltan Szallasi, Victoria Gómez-Abellán, María José López-Andreo, Marcos Gragera, Juan C Gómez-Fernández, Rubén López-Nicolás, Senena Corbalán-García.

Parkinson’s disease (PD) is a disabling neurodegenerative disorder in which multiple cell types, including dopaminergic and cholinergic neurons, are affected. The mechanisms of neurodegeneration in PD are not fully understood, limiting the development of therapies directed at disease-relevant molecular targets. C. elegans is a genetically tractable model system that can be used to disentangle disease mechanisms in complex diseases such as PD. Such mechanisms can be studied combining high-throughput molecular profiling technologies such as transcriptomics and metabolomics. However, the integrative analysis of multi-omics data in order to unravel disease mechanisms is a challenging task without advanced bioinformatics training. Galaxy, a widely-used resource for enabling bioinformatics analysis by the broad scientific community, has poor representation of multi-omics integration pipelines. We present the integrative analysis of gene expression and metabolite levels of a C. elegans PD model using GAIT-GM, a new Galaxy tool for multi-omics data analysis. Using GAIT-GM, we discovered an association between branched-chain amino acid metabolism and cholinergic neurons in the C. elegans PD model. An independent follow-up experiment uncovered cholinergic neurodegeneration in the C. elegans model that is consistent with cholinergic cell loss observed in PD. GAIT-GM is an easy to use Galaxy-based tool for generating novel testable hypotheses of disease mechanisms involving gene-metabolite relationships.

Lauren M. McIntyre, Francisco Huertas, Alison M. Morse, Rachel Kaletsky, Coleen T. Murphy, Vrinda Kalia, Gary W. Miller, Olexander Moskalenko, Ana Conesa & Danielle E. Mor.

The assessment of the safety of nano-biomedical products for patients is an essential prerequisite for their market authorization. However, it is also required to ensure the safety of the workers who may be unintentionally exposed to the nano-biomaterials (NBMs) in these medical applications during their synthesis, formulation into products and end-of-life processing and also of the medical professionals (e.g., nurses, doctors, dentists) using the products for treating patients. There is only a handful of workplace risk assessments focussing on NBMs used in medical applications. Our goal is to contribute to increasing the knowledge in this area by assessing the occupational risks of magnetite (Fe3O4) nanoparticles coated with PLGA-b-PEG-COOH used as contrast agent in magnetic resonance imaging (MRI) by applying the software-based Decision Support System (DSS) which was developed in the EU H2020 project BIORIMA.

V. Cazzagon, E. Giubilato, L. Pizzol, C. Ravagli, S. Doumett, G. Baldi, M. Blosi, A. Brunelli, C. Fito, F. Huertas, A. Marcomini, E. Semenzin, A. Zabeo, I. Zanoni, D. Hristozov.

PKCε is highly expressed in mast cells and plays a fundamental role in the antigen-triggered activation of the allergic reaction. Although its regulation by diacylglycerols has been described, its regulation by acidic phospholipids and how this regulation leads to the control of downstream vesicle secretion is barely known. Here, we used structural and evolutionary studies to find the molecular mechanism that explains the selectivity of the C1B domain of PKCε by Phosphatidic Acid (PA). This resided in a collection of Arg residues that form a specific rim on the outer surface of the C1B domain, around the diacylglycerol binding cleft. In RBL-2H3 cells, this basic rim allowed the kinase to respond specifically to phosphatidic acid signals that induced its translocation to the plasma membrane and subsequent activation. Further experiments in cells that overexpress PKCε and a mutant of the PA binding site, showed that PA-dependent PKCε activation increased vesicle degranulation in RBL-2H3 cells, and this correlated with increased SNAP23 phosphorylation. Over-expression of PKCε in these cells also induced an increase in the number of docked vesicles containing SNAP23, when stimulated with PA. This accumulation could be attributed to the stabilizing effect of phosphorylation on the formation of the SNARE complex, which ultimately led to increased release of content in the presence of Ca2+ during the fusion process. Therefore, these findings reinforce the importance of PA signaling in the activation of PKCε, which could be an important target to inhibit the exacerbated responses of these cells in the allergic reaction.

Emilio M. Serrano-López, David López-Martínez, Juan C. Gómez-Fernández, Antonio Luis Egea-Jiménez, Senena Corbalán-García.

During the last decade, the use of nanomaterials, due to their multiple utilities, has exponentially increased. Nanomaterials have unique properties such as a larger specific surface area and surface activity, which may result in health and environmental hazards different from those demonstrated by the same materials in bulk form. Besides, due to their small size, they can easily penetrate through the environmental and biological barriers. In terms of exposure potential, the vast majority of studies are focused on workplace areas, where inhalation is the most common route of exposure. The main route of entry into the environment is due to indirect emissions of nanomaterials from industrial settings, as well as uncontrollable releases into the environment during the use, recycling and disposal of nano-enabled products. Accidental spills during production or later transport of nanomaterials and release from wear and tear of materials containing nanomaterials may lead to potential exposure. In this sense, a proper understanding of all significant risks due to the exposure to nanomaterials that might result in a liability claim has been proved to be necessary. In this paper, the utility of an application for smartphones developed for the insurance sector has been validated as a solution for the analysis and evaluation of the emerging risk of the application of nanotechnology in the market. Different exposure scenarios for nanomaterials have been simulated with this application. The results obtained have been compared with real scenarios, corroborating that the use of novel tools can be used by companies that offer risk management in the form of insurance contracts.

Francisco Aznar Mollá,Carlos Fito-López, Jose Antonio Heredia Alvaro and Francisco Huertas-López.

Killer-cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) and effector T cells. Although KIR+ T cells accumulate in oncologic patients, their role in cancer immune response remains elusive. This study explored the role of KIR+CD8+ T cells in cancer immunosurveillance by analyzing their frequency at diagnosis in the blood of 249 patients (80 melanomas, 80 bladder cancers, and 89 ovarian cancers), their relationship with overall survival (OS) of patients, and their gene expression profiles. KIR2DL1+ CD8+ T cells expanded in the presence of HLA-C2-ligands in patients who survived, but it did not in patients who died. In contrast, presence of HLA-C1-ligands was associated with dose-dependent expansions of KIR2DL2/S2+ CD8+ T cells and with shorter OS. KIR interactions with their specific ligands profoundly impacted CD8+ T cell expression profiles, involving multiple signaling pathways, effector functions, the secretome, and consequently, the cellular microenvironment, which could impact their cancer immunosurveillance capacities. KIR2DL1/S1+ CD8+ T cells showed a gene expression signature related to efficient tumor immunosurveillance, whereas KIR2DL2/L3/S2+CD8+ T cells showed transcriptomic profiles related to suppressive anti-tumor responses. These results could be the basis for the discovery of new therapeutic targets so that the outcome of patients with cancer can be improved.

Lourdes Gimeno, Emilio M Serrano-López, José A Campillo, María A Cánovas-Zapata, Omar S Acuña, Francisco García-Cózar, María V Martínez-Sánchez, María D Martínez-Hernández, María F Soto-Ramírez, Pedro López-Cubillana, Jorge Martínez-Escribano, Jerónimo Martínez-García, Senena Corbalan-García, María R Álvarez-López, Alfredo Minguela.

In recent years, nanomaterials have gained tremendous attention due to their wide variety of industrial applications including food packaging, consumer products, nanomedicines, etc. The fascinating properties of nanoparticles which are responsible for creating several exciting opportunities, however, are also accountable for growing concerns of their toxic effects on humans as well as the environment. Thus, in the present study, the authors have developed generalized models for predicting the cytotoxicity and genotoxicity of seven metal oxide nanoparticles. The models not only take into account the structural features, but also the diverse experimental conditions under which the toxicity of nanoparticles was determined. The diverse experimental conditions were captured in the generalized models using the Box-Jenkins moving average approach. Here, two machine learning techniques, namely, linear discriminant analysis and random forest were utilized to build the final models. Importantly, the validation metrics showed that the developed models have significant discriminatory power.

Pravin Ambure, Arantxa Ballesteros, Francisco Huertas, Pau Camilleri, Stephen J. Barigye, Rafael Gozalbes.

Altered methylation patterns are driving forces in colorectal carcinogenesis. The serrated adenocarcinoma (SAC) and sporadic colorectal carcinoma showing histological and molecular features of microsatellite instability (hmMSI-H) are two endpoints of the so-called serrated pathological route sharing some characteristics but displaying a totally different immune response and clinical outcome. However, there are no studies comparing the methylome of these two subtypes of colorectal carcinomas. The methylation status of 450,000 CpG sites using the Infinium Human Methylation 450 BeadChip array was investigated in 48 colorectal specimens, including 39 SACs and 9 matched hmMSI-H. Microarray data comparing SAC and hmMSI-H showed an enrichment in functions related to morphogenesis, neurogenesis, cytoskeleton, metabolism, vesicle transport and immune response and also significant differential methylation of 1540 genes, including CD14 and HLA-DOA which were more methylated in hmMSI-H than in SAC and subsequently validated at the CpG, mRNA and protein level using pyrosequencing, quantitative polymerase chain reaction (qPCR) and immunohistochemistry. These results demonstrate particular epigenetic regulation patterns in SAC which may help to define key molecules responsible for the characteristic weak immune response of SAC and identify potential targets for treating SAC, which lacks molecular targeted therapy.

José García-Solano, María C. Turpin, Daniel Torres-Moreno, Francisco Huertas-López, Anne Tuomisto, Markus J. Mäkinen, Ana Conesa & Pablo Conesa-Zamora.

The intrinsic characteristics of the tumor microenvironment (TME), including acidic pH and overexpression of hydrolytic enzymes, offer an exciting opportunity for the rational design of TME-drug delivery systems (DDS). We developed and characterized a pH-responsive biodegradable poly-L-glutamic acid (PGA)-based combination conjugate family with the aim of optimizing anticancer effects. We obtained combination conjugates bearing Doxorubicin (Dox) and aminoglutethimide (AGM) with two Dox loadings and two different hydrazone pH-sensitive linkers that promote the specific release of Dox from the polymeric backbone within the TME. Low Dox loading coupled with a short hydrazone linker yielded optimal effects on primary tumor growth, lung metastasis (∼90% reduction), and toxicological profile in a preclinical metastatic triple-negative breast cancer (TNBC) murine model. The use of transcriptomic analysis helped us to identify the molecular mechanisms responsible for such results including a differential immunomodulation and cell death pathways among the conjugates. This data highlights the advantages of targeting the TME, the therapeutic value of polymer-based combination approaches, and the utility of –omics-based analysis to accelerate anticancer DDS.

Juan J. Arroyo-Crespo, Ana Armiñán, David Charbonnier, Leandro Balzano-Nogueira, Francisco Huertas-López, Cristina Martí, Sonia Tarazona, Jerónimo Forteza, Ana Conesa, María J. Vicent.