Adaptación de un sistema de propagación de priones in vitro para el cribado masivo de compuestos específicos frente a las Encefalopatías Espongiformes Transmisibles humanas

200 p.Las Encefalopatías Espongiformes Transmisibles (EET) son un conjunto de desórdenes neurodegenerativos mortales que afectan a varios mamíferos incluidos los humanos, cuyo agente causante es una isoforma anormalmente plegada (PrPSc) de la proteína del prion celular (PrPC) que adquiere propiedades neurotóxicas y la capacidad de inducir su conformación a otras proteínas del prion. Ninguna de las estrategias terapéuticas desarrolladas durante las últimas décadas ha sido efectiva frente a las EET humanas. La escasez de modelos in vitro e in vivo adecuados para el estudio de las EET humanas hacen que el cribado masivo de compuestos inhibidores de la propagación priónica sea una de las estrategias más exploradas en la actualidad. La mayoría de las moléculas con potencial terapéutico estudiadas en modelos no específicos frente a las EET humanas han revelado un problema de especificidad de especie e incluso de cepa priónica. En esta tesis doctoral, basándonos en la metodología de propagación de priones in vitro PMSA, y tras la modificación de los parámetros fundamentales, se han obtenido distintas PrP recombinantes humanas malplegadas espontáneamente con características depriones bona fide, cuya adaptación a un sistema de cribado masivo ha permitido evaluar la capacidad anti-priónica de un grupo de 18 tetrapirroles. A su vez se ha explorado la capacidad de adsorción de las PrP recombinantes humanas malplegadas a partículas esféricas, lo que ha permitido desarrollar una novedosa metodología de propagación sencilla, rápida y reproducible apta para el cribado masivo de compuestos. Así, los nuevos sistemas de cribado masivo de compuestos anti-priónicos han quedado validados para la búsqueda de compuestos terapéuticos específicos frente a las EET humanas

Adaptación de un sistema de propagación de priones in vitro para el cribado masivo de compuestos específicos frente a las Encefalopatías Espongiformes Transmisibles humanas

200 p.Las Encefalopatías Espongiformes Transmisibles (EET) son un conjunto de desórdenes neurodegenerativos mortales que afectan a varios mamíferos incluidos los humanos, cuyo agente causante es una isoforma anormalmente plegada (PrPSc) de la proteína del prion celular (PrPC) que adquiere propiedades neurotóxicas y la capacidad de inducir su conformación a otras proteínas del prion. Ninguna de las estrategias terapéuticas desarrolladas durante las últimas décadas ha sido efectiva frente a las EET humanas. La escasez de modelos in vitro e in vivo adecuados para el estudio de las EET humanas hacen que el cribado masivo de compuestos inhibidores de la propagación priónica sea una de las estrategias más exploradas en la actualidad. La mayoría de las moléculas con potencial terapéutico estudiadas en modelos no específicos frente a las EET humanas han revelado un problema de especificidad de especie e incluso de cepa priónica. En esta tesis doctoral, basándonos en la metodología de propagación de priones in vitro PMSA, y tras la modificación de los parámetros fundamentales, se han obtenido distintas PrP recombinantes humanas malplegadas espontáneamente con características depriones bona fide, cuya adaptación a un sistema de cribado masivo ha permitido evaluar la capacidad anti-priónica de un grupo de 18 tetrapirroles. A su vez se ha explorado la capacidad de adsorción de las PrP recombinantes humanas malplegadas a partículas esféricas, lo que ha permitido desarrollar una novedosa metodología de propagación sencilla, rápida y reproducible apta para el cribado masivo de compuestos. Así, los nuevos sistemas de cribado masivo de compuestos anti-priónicos han quedado validados para la búsqueda de compuestos terapéuticos específicos frente a las EET humanas

Mendler-style Iso-(Co)inductive predicates: a strongly normalizing approach

We present an extension of the second-order logic AF2 with iso-style inductive and coinductive definitions specifically designed to extract programs from proofs a la Krivine-Parigot by means of primitive (co)recursion principles. Our logic includes primitive constructors of least and greatest fixed points of predicate transformers, but contrary to the common approach, we do not restrict ourselves to positive operators to ensure monotonicity, instead we use the Mendler-style, motivated here by the concept of monotonization of an arbitrary operator on a complete lattice. We prove an adequacy theorem with respect to a realizability semantics based on saturated sets and saturated-valued functions and as a consequence we obtain the strong normalization property for the proof-term reduction, an important feature which is absent in previous related work.Comment: In Proceedings LSFA 2011, arXiv:1203.542

Association of acid phosphatase locus 1*C allele with the risk of cardiovascular events in rheumatoid arthritis patients

Introduction: Acid phosphatase locus 1 (ACP1) encodes a low molecular weight phosphotyrosine phosphatase implicated in a number of different biological functions in the cell. The aim of this study was to determine the contribution of ACP1 polymorphisms to susceptibility to rheumatoid arthritis (RA), as well as the potential contribution of these polymorphisms to the increased risk of cardiovascular disease (CV) observed in RA patients. Methods: A set of 1,603 Spanish RA patients and 1,877 healthy controls were included in the study. Information related to the presence/absence of CV events was obtained from 1,284 of these participants. All individuals were genotyped for four ACP1 single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247, and rs3828329, using a predesigned TaqMan SNP genotyping assay. Classical ACP1 alleles (*A, *B and *C) were imputed with SNP data. Results: No association between ACP1 gene polymorphisms and susceptibility to RA was observed. However, when RA patients were stratified according to the presence or absence of CV events, an association between rs11553742*T and CV events was found (P = 0.012, odds ratio (OR) = 2.62 (1.24 to 5.53)). Likewise, the ACP1*C allele showed evidence of association with CV events in patients with RA (P = 0.024, OR = 2.43). Conclusions: Our data show that the ACP1*C allele influences the risk of CV events in patients with R

Development of a New largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions

SARS-CoV-2-Mediated Lung Edema and Replication Are Diminished by Cystic Fibrosis Transmembrane Conductance Regulator Modulators

20 Pág.Coronaviruses (CoVs) of genera α, β, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.This work was supported by grants from the Government of Spain (BIO2016-75549-R; PID2019-107001RB-I00 AEI/FEDER, UE; SEV 2017-0712 and PIE_INTRAMURAL_LINEA 1- 202020E079), CSIC (PIE_INTRAMURAL-202020E043), the European Zoonotic Anticipation and Preparedness Initiative (ZAPI) (IMI_JU_115760), the European Commission (H2020-SC1- 2019, ISOLDA Project No. 848166-2), and the U.S. National Institutes of Health (NIH) (2P01AI060699). J.M.H. received a contract from Comunidad de Madrid (Y2020/BIO-6576, COVID-PREclinical-MODels-CM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In vivo experiments were performed at INIA-CISA (Madrid, Spain)Peer reviewe

A Large-Scale Genetic Analysis Reveals a Strong Contribution of the HLA Class II Region to Giant Cell Arteritis Susceptibility

We conducted a large-scale genetic analysis on giant cell arteritis (GCA), a polygenic immune-mediated vasculitis. A case-control cohort, comprising 1,651 case subjects with GCA and 15,306 unrelated control subjects from six different countries of European ancestry, was genotyped by the Immunochip array. We also imputed HLA data with a previously validated imputation method to perform a more comprehensive analysis of this genomic region. The strongest association signals were observed in the HLA region, with rs477515 representing the highest peak (p = 4.05 × 10−40, OR = 1.73). A multivariate model including class II amino acids of HLA-DRβ1 and HLA-DQα1 and one class I amino acid of HLA-B explained most of the HLA association with GCA, consistent with previously reported associations of classical HLA alleles like HLA-DRB1∗04. An omnibus test on polymorphic amino acid positions highlighted DRβ1 13 (p = 4.08 × 10−43) and HLA-DQα1 47 (p = 4.02 × 10−46), 56, and 76 (both p = 1.84 × 10−45) as relevant positions for disease susceptibility. Outside the HLA region, the most significant loci included PTPN22 (rs2476601, p = 1.73 × 10−6, OR = 1.38), LRRC32 (rs10160518, p = 4.39 × 10−6, OR = 1.20), and REL (rs115674477, p = 1.10 × 10−5, OR = 1.63). Our study provides evidence of a strong contribution of HLA class I and II molecules to susceptibility to GCA. In the non-HLA region, we confirmed a key role for the functional PTPN22 rs2476601 variant and proposed other putative risk loci for GCA involved in Th1, Th17, and Treg cell function

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Recombinant PrP and Its Contribution to Research on Transmissible Spongiform Encephalopathies

The misfolding of the cellular prion protein (PrPC) into the disease-associated isoform (PrPSc) and its accumulation as amyloid fibrils in the central nervous system is one of the central events in transmissible spongiform encephalopathies (TSEs). Due to the proteinaceous nature of the causal agent the molecular mechanisms of misfolding, interspecies transmission, neurotoxicity and strain phenomenon remain mostly ill-defined or unknown. Significant advances were made using in vivo and in cellula models, but the limitations of these, primarily due to their inherent complexity and the small amounts of PrPSc that can be obtained, gave rise to the necessity of new model systems. The production of recombinant PrP using E. coli and subsequent induction of misfolding to the aberrant isoform using different techniques paved the way for the development of cell-free systems that complement the previous models. The generation of the first infectious recombinant prion proteins with identical properties of brain-derived PrPSc increased the value of cell-free systems for research on TSEs. The versatility and ease of implementation of these models have made them invaluable for the study of the molecular mechanisms of prion formation and propagation, and have enabled improvements in diagnosis, high-throughput screening of putative anti-prion compounds and the design of novel therapeutic strategies. Here, we provide an overview of the resultant advances in the prion field due to the development of recombinant PrP and its use in cell-free systems