Desarrollo y caracterización de una línea mutante de pez cebra para auts2a, gen candidato a trastornos del espectro autista

La etiología multifactorial de los trastornos del espectro autista (TEA) dificulta en gran medida la comprensión de los mecanismos desencadenantes, por lo que investigar la implicación de genes candidatos es fundamental. El empleo de líneas mutantes de pez cebra ofrece información con gran potencial para ser trasladada a la práctica clínica. En esta tesis se ha desarrollado una línea mutante para auts2a, ortólogo del gen humano AUTS2, mediante CRISPR/Cas9, y se ha caracterizado a nivel fenotípico, celular y transcriptómico. Conocidas las alteraciones que la disrupción del gen provoca, se pretende establecer a auts2a como potencial diana terapéutica para la búsqueda de nuevos tratamientos y así implementar el consejo genético, lo que redundaría en una mejora de la calidad de vida de los pacientes y sus familiares

Modeling Cancer Using Zebrafish Xenografts: Drawbacks for Mimicking the Human Microenvironment

The first steps towards establishing xenografts in zebrafish embryos were performed by Lee et al., 2005 and Haldi et al., 2006, paving the way for studying human cancers using this animal species. Since then, the xenograft technique has been improved in different ways, ranging from optimizing the best temperature for xenografted embryo incubation, testing different sites for injection of human tumor cells, and even developing tools to study how the host interacts with the injected cells. Nonetheless, a standard protocol for performing xenografts has not been adopted across laboratories, and further research on the temperature, microenvironment of the tumor or the cell–host interactions inside of the embryo during xenografting is still needed. As a consequence, current non-uniform conditions could be affecting experimental results in terms of cell proliferation, invasion, or metastasis; or even overestimating the effects of some chemotherapeutic drugs on xenografted cells. In this review, we highlight and raise awareness regarding the different aspects of xenografting that need to be improved in order to mimic, in a more efficient way, the human tumor microenvironment, resulting in more robust and accurate in vivo resultsConsellería de Educación, Universidade e Formación Profesional (ED431C 2018/28)S

Zebrafish Models for the Safety and Therapeutic Testing of Nanoparticles with a Focus on Macrophages

Review[Abstract] New nanoparticles and biomaterials are increasingly being used in biomedical research for drug delivery, diagnostic applications, or vaccines, and they are also present in numerous commercial products, in the environment and workplaces. Thus, the evaluation of the safety and possible therapeutic application of these nanomaterials has become of foremost importance for the proper progress of nanotechnology. Due to economical and ethical issues, in vitro and in vivo methods are encouraged for the testing of new compounds and/or nanoparticles, however in vivo models are still needed. In this scenario, zebrafish (Danio rerio) has demonstrated potential for toxicological and pharmacological screenings. Zebrafish presents an innate immune system, from early developmental stages, with conserved macrophage phenotypes and functions with respect to humans. This fact, combined with the transparency of zebrafish, the availability of models with fluorescently labelled macrophages, as well as a broad variety of disease models offers great possibilities for the testing of new nanoparticles. Thus, with a particular focus on macrophage–nanoparticle interaction in vivo, here, we review the studies using zebrafish for toxicological and biodistribution testing of nanoparticles, and also the possibilities for their preclinical evaluation in various diseases, including cancer and autoimmune, neuroinflammatory, and infectious diseases.Xunta de Galicia; ED481A-2018/095A.P.-L. is supported by the Xunta de Galicia Pre-doctoral Fellowship (ED481A-2018/095); F.T.A. is recipient of a grant by the AECC (“Asociación Española Contra el Cáncer”, Spain). J.F.-R. was supported by a scholarship awarded by “Fundación Barrié”

Experimental Models to Study Autism Spectrum Disorders: hiPSCs, Rodents and Zebrafish

Autism Spectrum Disorders (ASD) affect around 1.5% of the global population, which manifest alterations in communication and socialization, as well as repetitive behaviors or restricted interests. ASD is a complex disorder with known environmental and genetic contributors; however, ASD etiology is far from being clear. In the past decades, many efforts have been put into developing new models to study ASD, both in vitro and in vivo. These models have a lot of potential to help to validate some of the previously associated risk factors to the development of the disorder, and to test new potential therapies that help to alleviate ASD symptoms. The present review is focused on the recent advances towards the generation of models for the study of ASD, which would be a useful tool to decipher the bases of the disorder, as well as to conduct drug screenings that hopefully lead to the identification of useful compounds to help patients deal with the symptoms of ASDConsellería de Educación, Universidade e Formación Profesional (ED431C 2018/28)//FIS PI19/00809 ISCIII//Xunta de Galicia (Centro singular de investigación de Galicia acreditación 2019–2022) and the European Union (European Regional Development Fund - ERDF) (ED431G 2019/02)S

Zebrafish as a platform to evaluate the potential of lipidic nanoemulsions for gene therapy in cancer

Gene therapy is a promising therapeutic approach that has experienced significant groth in recent decades, with gene nanomedicines reaching the clinics. However, it is still necessary to continue developing novel vectors able to carry, protect, and release the nucleic acids into the target cells, to respond to the widespread demand for new gene therapies to address current unmet clinical needs. We propose here the use of zebrafish embryos as an in vivo platform to evaluate the potential of newly developed nanosystems for gene therapy applications in cancer treatment. Zebrafish embryos have several advantages such as low maintenance costs, transparency, robustness, and a high homology with the human genome. In this work, a new type of putrescine-sphingomyelin nanosystems (PSN), specifically designed for cancer gene therapy applications, was successfully characterized and demonstrated its potential for delivery of plasmid DNA (pDNA) and miRNA (miR). On one hand, we were able to validate a regulatory effect of the PSN/miR on gene expression after injection in embryos of 0 hpf. Additionally, experiments proved the potential of the model to study the transport of the associated nucleic acids (pDNA and miR) upon incubation in zebrafish water. The biodistribution of PSN/pDNA and PSN/miR in vivo was also assessed after microinjection into the zebrafish vasculature, demonstrating that the nucleic acids remained associated with the PSN in an in vivo environment, and could successfully reach disseminated cancer cells in zebrafish xenografts. Altogether, these results demonstrate the potential of zebrafish as an in vivo model to evaluate nanotechnology-based gene therapies for cancer treatment, as well as the capacity of the developed versatile PSN formulation for gene therapy applicationsThis research was funded by the Instituto de Salud Carlos IIIISCIII and the European Regional Development Fund (FEDER) (AC18/00107, AC18/00045, PI18/00176); by the ERA-NET EURONANOMED III project METASTARG (Grant Number JTC 2018-045) and the ERA-NET EURONANOMED III project PANIPAC (Grant Number JTC 2018/041); and by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria (IN607B2021/14). RP was supported by Roche-Chus Joint Unit (IN853B 2018/03) funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria. SL, PH, and AP-L. were funded by a Predoctoral fellowship (IN606A-2019/003, IN606A-2018/019 and ED481A-2018/095) from Axencia Galega de Innovación (GAIN, Xunta de Galicia).S

Cellular and Molecular Mechanisms Underlying Glioblastoma and Zebrafish Models for the Discovery of New Treatments

Glioblastoma (GBM) is the most common of all brain malignant tumors; it displays a median survival of 14.6 months with current complete standard treatment. High heterogeneity, aggressive and invasive behavior, the impossibility of completing tumor resection, limitations for drug administration and therapeutic resistance to current treatments are the main problems presented by this pathology. In recent years, our knowledge of GBM physiopathology has advanced significantly, generating relevant information on the cellular heterogeneity of GBM tumors, including cancer and immune cells such as macrophages/microglia, genetic, epigenetic and metabolic alterations, comprising changes in miRNA expression. In this scenario, the zebrafish has arisen as a promising animal model to progress further due to its unique characteristics, such as transparency, ease of genetic manipulation, ethical and economic advantages and also conservation of the major brain regions and blood–brain–barrier (BBB) which are similar to a human structure. A few papers described in this review, using genetic and xenotransplantation zebrafish models have been used to study GBM as well as to test the anti-tumoral efficacy of new drugs, their ability to interact with target cells, modulate the tumor microenvironment, cross the BBB and/or their toxicity. Prospective studies following these lines of research may lead to a better diagnosis, prognosis and treatment of patients with GBMF.T.A. has been supported by the AECC (“Asociación Española Contra el Cáncer”, Spain). We would also like to thank the following: the Talento Program from Madrid Government, Spain (2017-T1/BMD-5333); Convocatoria 2018 de proyectos de I+D+i «RETOS INVESTIGACIÓN» (RTI2018-095061-B-I00) (to C.M.R.); “Convocatoria de ayudas para la contratación de ayudantes de investigación” (PEJ-2018-AI/BMD-9724) (to M.T.-P.); the Xunta de Galicia Pre-doctoral Fellowship (ED481A-2018/095) (to A.P.L.)S

Edelfosine nanoemulsions inhibit tumor growth of triple negative breast cancer in zebrafish xenograft model

Triple negative breast cancer (TNBC) is known for being very aggressive, heterogeneous and highly metastatic. The standard of care treatment is still chemotherapy, with adjacent toxicity and low efficacy, highlighting the need for alternative and more effective therapeutic strategies. Edelfosine, an alkyl-lysophospholipid, has proved to be a promising therapy for several cancer types, upon delivery in lipid nanoparticles. Therefore, the objective of this work was to explore the potential of edelfosine for the treatment of TNBC. Edelfosine nanoemulsions (ET-NEs) composed by edelfosine, Miglyol 812 and phosphatidylcholine as excipients, due to their good safety profile, presented an average size of about 120 nm and a neutral zeta potential, and were stable in biorelevant media. The ability of ET-NEs to interrupt tumor growth in TNBC was demonstrated both in vitro, using a highly aggressive and invasive TNBC cell line, and in vivo, using zebrafish embryos. Importantly, ET-NEs were able to penetrate through the skin barrier of MDA-MB 231 xenografted zebrafish embryos, into the yolk sac, leading to an effective decrease of highly aggressive and invasive tumoral cells’ proliferation. Altogether the results demonstrate the potential of ET-NEs for the development of new therapeutic approaches for TNBCThis work was in part supported by grants from Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund (FEDER) (PI18/00176; FI19/00206), the Axencia Galega de Innovación, Conselleria de Educación, Universidade e Formación profesional (IN606A-2019/003; ED431C 2018/28; ED481A-2018/095) and the Spanish Ministry of Education, Culture, and Sport (FPU15/06595).S

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort

Background Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster.Methods Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3.Results Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3.Conclusions During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis

Morphological Abnormalities and Gene Expression Changes Caused by High Incubation Temperatures in Zebrafish Xenografts with Human Cancer Cells

Published studies show that most of the human cancer xenograft studies in zebrafish embryos have used incubation temperatures in the range of 32–34 °C for 3–6 days post-injection, trying to find a compromise temperature between the zebrafish embryos (28 °C) and the human injected cells (37 °C). While this experimental setup is widely used, a question remains: is possible to overcome the drawbacks caused by a suboptimal temperature for the injected cells? To clarify the effect of temperature and injected cells on the host, in this study, we analyzed the development and health of the last in response to different temperatures in the presence or absence of injected human cancer cells. Comparing different incubation temperatures (28, 34 and 36 °C), we determined morphological abnormalities and developmental effects in injected and non-injected embryos at different time points. Besides this, the expression of selected genes was determined by qPCR to determine temperature affected metabolic processes in the embryos. The results indicate that an incubation temperature of 36 °C during a period of 48 h is suitable for xenotransplantation without morphological or metabolic changes that could be affecting the host or the injected cells, allowing them to proliferate near their optimal temperatureThis research is funded by ‘Consolidación e estruturación de unidades de investigación competitivas do SUG. Grupos de referencia competitiva’ (ED431C 2018/29) and ‘Axuda á formación da etapa predoutoral’ of Xunta de GaliciaS