Expresión y función de los factores de transcripción FoxE1 y Sox9 en la célula folicular tiroidea

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 14-07-2017Esta tesis tiene embargado el acceso al texto completo hasta el 14-01-201

New insights into FoxE1 functions: identification of direct FoxE1 targets in thyroid cells

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: FoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility. [Methodology/Principal Finding]:I n order to further investigate downstream FoxE1 targets, we performed a genome-wide expression screening after knocking-down FoxE1 and obtained new insights into FoxE1 transcriptional networks in thyroid follicular cells. After validation, we confirmed Adamts9, Cdh1, Duox2 and S100a4 as upregulated genes and Casp4, Creld2, Dusp5, Etv5, Hsp5a, Nr4a2 and Tm4sf1 as downregulated genes when FoxE1 was silenced. In promoter regions of putative FoxE1-regulated genes and also in the promoters of the classical thyroid genes Nis, Pax8 and Titf1, we performed an in silico search of the FoxE1 binding motif that was in close proximity to the NF1/CTF binding sequence, as previously described for other forkhead factors. Using chromatin immunoprecipitation we detected specific in vivo FoxE1 binding to novel regulatory regions in two relevant thyroid genes, Nis and Duox2. Moreover, we demonstrated simultaneous binding of FoxE1 and NF1/CTF to the Nis upstream enhancer region, as well as a clear functional activation of the Nis promoter by both transcription factors. [Conclusions/Significance]:In search for potential downstream mediators of FoxE1 function in thyroid cells, we identified two novel direct FoxE1 target genes. To our knowledge, this is the first evidence regarding the implication of Nis and Duox2 in executing the transcriptional program triggered by FoxE1. Furthermore, this study points out the important role of FoxE1 in the regulation of a large number of genes in thyroid cells. © 2013 Fernández et al.This work was supported by Grants BFU-2010-16025 from the Dirección General de Proyectos de Investigación; RD06/0020/0060 and RD12/0036/0030 from FIS, Instituto de Salud Carlos III; and S2011/BMD-2328 TIRONET project from the Comunidad de Madrid (Spain). LP Fernández holds a postdoctoral grant of the Juan de la Cierva programme of the Spanish Government.Peer Reviewe

Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells

The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defectThis book is based upon work from COST Action DARTER (CA 17103), supported by COST (European Cooperation in Science and Technology)

Ampicillin plus ceftriaxone combined therapy for enterococcus faecalis infective endocarditis in opat

Ampicillin plus ceftriaxone (AC) is a well-recognized inpatient regimen for Enterococcus faecalis infective endocarditis (IE). In this regimen, ceftriaxone is usually administered 2 g every 2 h (AC12). The administration of AC in outpatient parenteral antibiotic treatment (OPAT) programs is challenging because multiple daily doses are required. AC regimens useful for OPAT programs include once-daily high-dose administration of ceftriaxone (AC24) or AC co-diluted and jointly administered in bolus every 4 h (ACjoined). In this retrospective analysis of prospectively collected cases, we aimed to assess the clinical effectivity and safety of three AC regimens for the treatment of E. faecalis IE. Fifty-nine patients were treated with AC combinations (AC12 n = 32, AC24 n = 17, and ACjoined n = 10). Six relapses occurred in the whole cohort: five (29.4%) treated with AC24 regimen and one (10.0%) with ACjoined. Patients were cured in 30 (93.3%), 16 (94.1%), and eight (80.0%) cases in the AC12, AC24 and ACjoined groups, respectively. Unplanned readmission occurred in eight (25.0%), six (35.3%), and two (20.0%) patients in the AC12, AC24 and ACjoined groups, respectively. The outcome of patients with E. faecalis IE treated with AC in OPAT programs relies on an optimization of the delivery of the combination. AC24 exhibit an unexpected rate of failures, however, ACjoined might be an effective alternative which clinical results should corroborate in further studies

Generation and characterization of a human iPSC line (UAMi005-A) from a patient with nonketotic hyperglycinemia due to mutations in the GLDC gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discoveryResearch reported in this work was funded by Grants of Spanish Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (FEDER) PI16/00573, and Fundación Isabel Gemio- Fundación La Caixa (LCF/PR/PR16/11110018). The authors thank the Cytogenetic unit from Centro Nacional de Investigaciones Oncológicas (CNIO) for its excellent technical assistance. Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces. LAC is a PhD student funded by the Asociación Española para el Estudio de Metabolopatías Congénitas (AEPMEC). ALM is a postdoctoral researcher of Comunidad Autónoma de Madrid (PEJD-2017-POST/BMD-3671). EAB is a PhD student funded by the FPU program of the Spanish Ministry of Science, Innovation and Universities (FPU15/02923

Ampicillin Plus Ceftriaxone Combined Therapy for Enterococcus faecalis Infective Endocarditis in OPAT

Cardiovascular Infectious Study Group of the Andalusian Society of Infectious Diseases.Ampicillin plus ceftriaxone (AC) is a well-recognized inpatient regimen for Enterococcus faecalis infective endocarditis (IE). In this regimen, ceftriaxone is usually administered 2 g every 2 h (AC12). The administration of AC in outpatient parenteral antibiotic treatment (OPAT) programs is challenging because multiple daily doses are required. AC regimens useful for OPAT programs include once-daily high-dose administration of ceftriaxone (AC24) or AC co-diluted and jointly administered in bolus every 4 h (ACjoined). In this retrospective analysis of prospectively collected cases, we aimed to assess the clinical effectivity and safety of three AC regimens for the treatment of E. faecalis IE. Fifty-nine patients were treated with AC combinations (AC12 n = 32, AC24 n = 17, and ACjoined n = 10). Six relapses occurred in the whole cohort: five (29.4%) treated with AC24 regimen and one (10.0%) with ACjoined. Patients were cured in 30 (93.3%), 16 (94.1%), and eight (80.0%) cases in the AC12, AC24 and ACjoined groups, respectively. Unplanned readmission occurred in eight (25.0%), six (35.3%), and two (20.0%) patients in the AC12, AC24 and ACjoined groups, respectively. The outcome of patients with E. faecalis IE treated with AC in OPAT programs relies on an optimization of the delivery of the combination. AC24 exhibit an unexpected rate of failures, however, ACjoined might be an effective alternative which clinical results should corroborate in further studies.The authors received no financial support for the research, authorship, and/or publication of this article. GVA was supported by the Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (“A way to achieve Europe”), Subprograma Miguel Servet (grant CP19/00159). HHL was supported by the Instituto de Salud Carlos III, Subprograma Rio Hortega (grant CM19/00152)

Blood culture-negative infective endocarditis: a worse outcome? Results from a large multicentre retrospective Spanish cohort study

[Background] To assess the impact of blood cultures negative infective endocarditis (BCNIE) on in-hospital mortality.[Methods] Prospective multicentre study with retrospective analysis of a Spanish cohort including adult patients with definite IE. Cardiac implantable devices infection were excluded. Comparisons between blood cultures positive and BCNIE groups were performed to analyse in-hospital mortality.[Results] 1001 cases were included of which 83 (8.3%) had BCNIE. Alternative microbiological diagnosis was achieved for 39 (47%) out 83 cases. The most frequent identifications were: Coxiella burnetii (11; 28.2%), Tropheryma whipplei (4; 10.3%), Streptococcus gallolyticus (4;10.3%) and Staphylococcus epidermidis (3; 7.7%). Surgery was performed more frequently in BCNIE group (57.8 vs. 36.9%, p < .001). All-cause in-hospital mortality rate was 26.7% without statistical difference between compared groups. BCNIE was not associated to worse mortality rate in Cox regression model (aHR = 1.37, 95% CI 0.90–2.07, p = .14). Absence of microbiological diagnosis was also not associated to worse in-hospital prognosis (aHR = 1.62, 95% CI 0.99–2.64, p = .06).[Conclusions] In our cohort, BCNIE was not associated to greater in-hospital mortality based in multivariate Cox regression models. The variables most frequently associated with mortality were indicated but not performed surgery (aHR = 2.48, 95% CI 1.73–3.56, p < .001), septic shock (aHR = 2.24, 95% CI 1.68–2.99, p < .001), age over 65 years (aHR = 1.88, 95% CI 1.40-2.52, p < .001) and complicated endocarditis (aHR = 1.79, 95% CI 1.36–2.37, p < .001).Peer reviewe

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Expresión y función de los factores de transcripción FoxE1 y Sox9 en la célula folicular tiroidea

Memoria de Tesis Doctoral presentada por Arístides López Márquez, Licenciado en Bioquímica para optar al grado de Doctor por la Universidad Autónoma de Madrid. Este trabajo ha sido realizado en el laboratorio de la profesora Pilar Santisteban Sanz en el Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM).[ES] Las células foliculares son el tipo celular mayoritario de la glándula tiroides y expresan las proteínas necesarias para la síntesis y secreción de las hormonas tiroideas, como el Simportador de Sodio/Iodo (NIS), la Tiroperoxidasa (TPO) o la Tiroglobulina (Tg). La expresión de estos marcadores de diferenciación tiroideos está regulada principalmente por TSH, IGF1 y TGFβ a través de la expresión y la activación de los factores de transcripción Nkx2-1, Pax8 y FoxE1. La expresión conjunta de estos tres factores tiene lugar exclusivamente en el tiroides desde el inicio de la especificación de los precursores tiroideos en el endodermo faríngeo hasta el tiroides adulto. Aunque se conocen algunas de las vías de señalización y los factores de transcripción implicados en la especificación y diferenciación de las células foliculares tiroideas, aún no se han descifrado los mecanismos subyacentes. Así mismo son desconocidos otros factores de transcripción implicados en esa compleja red transcripcional. Por ese motivo nos propusimos en primer lugar estudiar el papel de las vías de señalización de TSH y TGFβ en la regulación de la expresión de FoxE1, como uno de los factores de transcripción esencial en el desarrollo y la diferenciación tiroidea. Además, hemos estudiado la expresión y función en el tiroides de Sox9, factor de transcripción implicado en el desarrollo y diferenciación de órganos de origen endodérmico, como un posible candidato a regular la expresión de los factores de transcripción tiroideos en la especificación y diferenciación de la célula folicular tiroidea. En esta tesis hemos descrito que Sox9 se expresa en las células foliculares del tiroides embrionario concomitantemente a la expresión de los factores de transcripción tiroideos, manteniéndose su expresión durante el desarrollo embrionario hasta el tiroides adulto. Hemos identificado sitios de unión para los factores de transcripción tiroideos Nkx2-1, Pax8 y FoxE1 así como para Smads, Sox9 y sitios CRE en los promotores de FoxE1 y Sox9. Además, hemos demostrado que la TSH induce la expresión de FoxE1 y Sox9 a través de la vía del cAMP/PKA/CREB. El TGFβ, a través de las Smads, inhibe el efecto inductor de la TSH sobre la expresión de FoxE1 y Sox9. Así mismo nuestros datos muestran que la transcripción de FoxE1 y Sox9 está regulada por Nkx2-1, Pax8 y FoxE1 a través de la unión a sus respectivos promotores. Aunque está bien aceptado que FoxE1 regula la expresión de TPO y Tg, no se había reportado que dicho factor regulase a NIS. Nuestro estudio ha identificado un sitio de unión de FoxE1 en el promotor de NIS y hemos demostrado su funcionalidad, concluyendo que FoxE1 induce también la expresión de este gen de diferenciación tiroidea. Además, hemos demostrado que Sox9 se une al promotor de FoxE1 y al suyo propio regulando la expresión de ambos genes, confirmando la existencia de circuitos regulatorios transcripcionales en la diferenciación tiroidea. Esta tesis pone de manifiesto la implicación de Sox9 en la diferenciación de la célula folicular y aporta otra vía de estudio sobre la función de nuevos factores de transcripción. Además se ha avanzado en la identificación de nuevas dianas transcripcionales de FoxE1 y en la función de las vías de TSH y TGFβ, profundizando en el conocimiento de la regulación de la diferenciación de la célula folicular tiroidea.[EN] The follicular cells are the major cell type of the thyroid gland and express the necessary proteins for the synthesis and secretion of thyroid hormones, such as the Sodium/Iodine Symporter (NIS), Thyroperoxidase (TPO) or Thyroglobulin (Tg). The expression of these differentiation markers is mainly regulated by TSH, IGF1 and TGFβ through the expression and activation of the transcription factors Nkx2-1, Pax8 and FoxE1. The combined expression of these three factors occurs exclusively in the thyroid from the beginning of the specification of thyroid precursors in the pharyngeal endoderm to the adult thyroid. Although some of the signaling pathways and the transcription factors involved in the specification and differentiation of thyroid follicular cells are known, the underlying mechanisms have not yet been deciphered. Also other transcription factors involved in that complex transcriptional network are still unknown. For this reason we proposed to study the role of TSH and TGFβ signaling pathways in the regulation of FoxE1 expression as one of the essential transcription factors in thyroid development and differentiation. In addition, we have studied the expression and function in the thyroid of Sox9, a transcription factor involved in the development and differentiation of endodermal-derived-organs, as a possible candidate to regulate the expression of thyroid transcription factors in the specification and differentiation of thyroid follicular cell. In this thesis we have described that Sox9 is expressed in the follicular cells of the embryonic thyroid concomitantly with the expression of the thyroid transcription factors, maintaining its expression during the embryonic development up to the adult thyroid. We have identified binding sites for the thyroid transcription factors Nkx2-1, Pax8 and FoxE1 as well as for Smads, Sox9 and CRE sites in the promoters of FoxE1 and Sox9. In addition, we have demonstrated that TSH induces FoxE1 and Sox9 expression through the cAMP/PKA/CREB pathway. TGFβ, through the Smads, inhibits the TSH-inducedFoxE1 and Sox9 expression. Also our data show that the transcription of FoxE1 and Sox9 is regulated by Nkx2-1, Pax8 and FoxE1 through binding to their respective promoters. Although it is well accepted that FoxE1 regulates the expression of TPO and Tg, it has not been reported its role in NIS regulation. Our study has identified a FoxE1 binding site in the NIS promoter and we have demonstrated its functionality by concluding that FoxE1 also induces the expression of this thyroid differentiation gene. Furthermore, we have demonstrated that Sox9 binds to the FoxE1 promoter and to its own, regulating the expression of both genes and confirming the existence of transcriptional regulatory circuits in thyroid differentiation. This thesis demonstrates the implication of Sox9 in the differentiation of the thyroid follicular cell and provides another approach to study the function of new transcription factors. In addition, we have progressed in the identification of novel transcriptional FoxE1 targets and in the function of the TSH and TGFβ pathways, deepening the knowledge of the regulation of thyroid follicular cell differentiation.Este trabajo ha sido financiado por una Beca de Formación de Personal Investigador (FPI) de la Universidad Autónoma de Madrid.Peer reviewe

Hormonal regulation of FoxE1 expression in thyroid epithelial cells

Resumen del póster presentado al 38th Annual Meeting of the European Thyroid Association celebrado en Santiago de Compostela (España) del 6 al 10 de septiembre de 2014.Differentiation of thyroid follicular cells is regulated by hormones and growth factors. Among these signals, TSH, IGF1 and TGF play a predominant role. These three ligands transcriptionally regulate markers of thyroid differentiation such as Thyroglobulin (Tg) Thyroperoxidase (TPO) and sodium iodide symporter (NIS). This effect takes place, at least in part, through a responsive element to where the FoxE1 binds, opening chromatin and creating an environment that facilitate the binding of other transcription factors. Taking in consideration the proposed role of FoxE1 as a mediator of the hormone/growth factor transcriptional action, the main objective of this work was to study the regulation of FoxE1 expression by TSH, IGF1 and TGFβ. Mouse FoxE1 promoter was cloned and transient transfections were performed in PCCL3 in presence or absence of TSH, IGF1 and TGFβ. Forskolin and H89 were used to study the cAMP/PKA pathway. Co-transfections in Hela cells were performed to elucidate the role of other transcription factors. FoxE1 protein and mRNA levels in different conditions were evaluated by westernblot and RT-qPCR. The in silico analysis of FoxE1 promoter reveals the presence of a CRE and several Smad binding sites in its sequence. TSH and IGF1 induce the transcription of FoxE1 by the activation of its promoter. The TSH effect is cAMP/ PKA dependent and therefore CREB positively regulates FoxE1 transcription. IGF1 cooperates with TSH while TGF inhibits the TSH induction on FoxE1 gene expression. Not effect was found of TGF on IGF1 action. We conclude that there is a complex network of signals and transcription factors involved in FoxE1 expression that are essential to maintain a fine regulation of this important gene that control thyroid cell differentiation.Peer reviewe