Papel del componente 3 del complemento (C3) como mediador de C/EBPβ en los procesos inflamatorios y excitotóxicos del sistema nervioso central

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 27 de marzo, 2014CCAAT/Enhancer-binding protein (C/EBP) es un factor de transcripción que inicialmente fue identificado como regulador de procesos de diferenciación e inflamación principalmente en tejido adiposo e hígado. Sin embargo, su función en el cerebro ha sido descrita más recientemente. Estudios previos de nuestro laboratorio revelaron que C/EBPβ estaba implicada en procesos de inflamación y daño cerebral, y que los ratones carentes de este gen eran menos susceptibles al daño excitotóxico inducido por ácido kaínico. Con el fin de analizar los mecanismos a través de los cuales C/EBPβ regula estos procesos, lo primero que hicimos fue un análisis de microarrays con RNA aislado de hipocampo de ratones C/EBP +/+ y C/EBP -/-. Algunos de los genes expresados diferencialmente, eran genes implicados en inflamación y daño cerebral. Uno de esos genes es el del componente 3 del complemento (C3), cuya expresión aumenta en los ratones C/EBPβ+/+ y que está implicado en diversos desórdenes cerebrales. En este trabajo nos hemos centrado en el posible papel de C3 como mediador de C/EBP tras un daño excitotóxico e inflamatorio. Estudios de transfección con el promotor de C3 y estudios in vitro de daño neural, mostraron que C/EBPβ regula directamente la expresión de C3 tras un daño excitotóxico e inflamatorio. En este trabajo mostramos también que la reducción de C3 mediante RNA de interferencia, reduce la producción de agentes proinflamatorios como IL-1β y COX-2, así como la de nitritos en cultivos primarios de astrocitos tratados con lipopolisacárido. Usando un modelo in vivo de excitotoxicidad mostramos que ambas proteínas aumentan principalmente en la región CA3 del hipocampo. Por último, demostramos que ratones carentes de C/EBPβ presentan una importante reducción de la expresión de C3 tras la inyección de ácido kaínico. Estos resultados sugieren que C/EBPβ podría estar implicada en enfermedades neurológicas vinculadas a procesos inflamatorios y excitotóxicos, al menos en parte a través de la regulación directa de C3.The CCAAT/Enhancer-binding protein (C/EBP) is a transcription factor, which was first identified as a regulator of differentiation and inflammatory processes mainly in adipose tissue and liver; however its function in the brain was largely unknown for many years. Previous studies from our laboratory indicated that C/EBP is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. In order to further analyze the mechanisms by which C/EBP was involved in these processes, we first performed cDNA microarrays analysis using hippocampal RNA isolated from C/EBP +/+ and C/EBP -/- mice. Among the genes displaying significant changes in expression, some of them were involved in inflammatory processes and brain injury. One of those genes which expression was increased in C/EBPβ+/+ animals was complement component 3 (C3), a gene implicated in different brain disorders. This work has been focused in the possible involvement of C3 in the effects of C/EBPβ in excitotoxic injury. Transfection studies using C3 promoter and in vitro studies of neural damage, demonstrated that C/EBPβ directly regulates the expression of C3 following an excitotoxic and inflammatory injuries. In addition, we show that down-regulation of C3 by siRNA reduces proinflammatory molecules such us IL-1β and COX-2 and leads to a decrease of nitrite levels in an astrocyte culture after lipopolysaccharide treatment. By using an in vivo model of excitotocicity we demonstrate an increase of C/EBP and C3 in the CA3 subfield of the hippocampus. Finally, we show that mice lacking C/EBP exhibit a marked reduction in C3 expression after kainic acid injection. These results suggest that C/EBPβ could regulate brain disorders, in which excitotocic and inflammatory processes are involved, at least in part through the direct regulation of C3

Induction of Lysosome Membrane Permeabilization as a Therapeutic Strategy to Target Pancreatic Cancer Stem Cells

Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.Acknowledgments: This work was supported by MINECO (SAF2017-89643-R, SAF2014-57243-R, SAF2015-62547-ERC) (M.V.), Fundacion FERO (IX FERO Grant for Research in Oncology) (M.V.), Fundacio La Marato de TV3 (141) (M.V.), Melanoma Research Alliance (Bristol-Myers Squibb-Melanoma Research Alliance Young Investigator Award 2017 (https://doi.org/10.48050/pc.gr.75716)) (M.V.), Beug Foundation (Prize for Metastasis Research 2017) (M.V.), Fundacion Ramon Areces (CIVP19S8163) (M.V.) and CIVP20S10662 (E.O.P.), Worldwide Cancer Research (19-0177) (M.V.), H2020-FETOPEN (828972) (M.V.), Cancer Research Institute (Clinic and Laboratory Integration Program CRI Award 2018 (54545)) (M.V.), AECC (Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.), LAB AECC 2019 (LABAE19002-VALI) (M.V.), ERC CoG (864759) (M.V.), Sophien-Stiftung zur Förderung der klinischen Krebsforschung (T.W.), Promedica Stiftung (T.W.), Stiftung f€ur angewandte Krebsforschung (T.W.), Forschungskredit of the University of Zurich (FK-18-054) (T.W.), Betty and David Koetser Foundation for Brain Research (T.W.), Foundation for Applied Cancer Research in Zurich (T.W., M.W.), Comunidad de Madrid (S2017/BMD-3867 RENIM-CM and Y2018/NMT-4949 NanoLiver-CM) and European structural and investment funds (M.D.), ISCIII (PT20/00044) co-funded by FEDER “A way of making Europe” (M.D.), Ministero dell’Istruzione, dell’Universita e della Ricerca-MIUR, “Dipartimenti di Eccellenza 2018-2022”, (D15D18000410001) (L.B. and P.C.), Science Foundation Ireland Frontiers for the Future Award (19/FFP/6443) (L.Y.), Science Foundation Ireland Strategic Partnership Programme, Precision Oncology Ireland (18/SPP/3522) (L.Y.), Breast Cancer Now Fellowship Award/ with the generous support of Walk the Walk (2019AugSF1310) (D.V.), La Caixa-Severo Ochoa International PhD Program Fellowship (LCF/BQ/SO16/52270014) (L.Z.), La Caixa International PhD Program Fellowship-Marie Sklodowska-Curie (LCF/BQ/DI17/11620028) (P.G-G), MINECO-Severo Ochoa PhD Fellowship (BES-2017-081995) (L.A-E.), AECC Postdoctoral Fellowship (POSTD19016PRIE) (N.P.), Boehringer Ingelheim Fonds MD fellowship (L.M.). The contribution of the Experimental Therapeutics Programme was supported by core funding from the Spanish National Cancer Research Center (CNIO). CNIO is supported by the ISCIII, the Ministerio de Ciencia e Innovacion, and is a Severo Ochoa Center of Excellence (SEV-2015-0510). The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovacion and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.V. was named Ramon y Cajal Investigator (RYC-2013-13365) and is member of EMBO YIP (4053)

Multiple myeloma and SARS-CoV-2 infection: clinical characteristics and prognostic factors of inpatient mortality

There is limited information on the characteristics, prognostic factors, and outcomes of patients with multiplemyeloma (MM) hospitalized with COVID-19. This retrospective case series investigated 167 patients reported from 73hospitals within the Spanish Myeloma Collaborative Group network in March and April, 2020. Outcomes werecompared with 167 randomly selected, contemporary, age-/sex-matched noncancer patients with COVID-19 admittedat six participating hospitals. Among MM and noncancer patients, median age was 71 years, and 57% of patients weremale; 75 and 77% of patients, respectively, had at least one comorbidity. COVID-19 clinical severity wasmoderate-severe in 77 and 89% of patients and critical in 8 and 4%, respectively. Supplemental oxygen was requiredby 47 and 55% of MM and noncancer patients, respectively, and 21%/9% vs 8%/6% required noninvasive/invasiveventilation. Inpatient mortality was 34 and 23% in MM and noncancer patients, respectively. Among MM patients,inpatient mortality was 41% in males, 42% in patients aged >65 years, 49% in patients with active/progressive MM athospitalization, and 59% in patients with comorbid renal disease at hospitalization, which were independentprognostic factors on adjusted multivariate analysis. This case series demonstrates the increased risk and identifiespredictors of inpatient mortality among MM patients hospitalized with COVID-19

MM, SARS-CoV-2 infection, and inpatient mortality

There is limited information on the characteristics, pre-admission prognostic factors, and outcomes of patients with multiple myeloma (MM) hospitalized with coronavirus disease 2019 (COVID-19). This retrospective case series investigated characteristics and outcomes of 167 MM patients hospitalized with COVID-19 reported from 73 hospitals within the Spanish Myeloma Collaborative Group network in Spain between March 1 and April 30, 2020. Outcomes were compared with a randomly selected contemporary cohort of 167 age-/sex-matched non-cancer patients with COVID-19 admitted at 6 participating hospitals. Common demographic, clinical, laboratory, treatment, and outcome variables were collected; specific disease status and treatment data were collected for MM patients. Among the MM and non-cancer patients, median age was 71 years and 57% of patients were male in each series, and 75% and 77% of patients, respectively, had at least one comorbidity. COVID-19 clinical severity was moderate-severe in 77% and 89% of patients and critical in 8% and 4%, respectively. Supplemental oxygen was required by 47% and 55% of MM and non-cancer patients, respectively, and 21%/9% vs 8%/6% required non-invasive/invasive ventilation. Inpatient mortality was 34% and 23% in MM and non-cancer patients, respectively. Among MM patients, inpatient mortality was 41% in males, 42% in patients aged >65 years, 49% in patients with active/progressive MM at hospitalization, and 59% in patients with comorbid renal disease at hospitalization, which were independent prognostic factors of inpatient mortality on adjusted multivariate analysis. This case series demonstrates the increased risk and identifies predictors of inpatient mortality among MM patients hospitalized with COVID-19.This study was supported by PETHEMA FoundationN

A clinically compatible drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

We report a medium‐throughput drug‐screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood–brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug‐screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere

Multiple myeloma and SARS-CoV-2 infection: clinical characteristics and prognostic factors of inpatient mortality

There is limited information on the characteristics, prognostic factors, and outcomes of patients with multiple myeloma (MM) hospitalized with COVID-19. This retrospective case series investigated 167 patients reported from 73 hospitals within the Spanish Myeloma Collaborative Group network in March and April, 2020. Outcomes were compared with 167 randomly selected, contemporary, age-/sex-matched noncancer patients with COVID-19 admitted at six participating hospitals. Among MM and noncancer patients, median age was 71 years, and 57% of patients were male; 75 and 77% of patients, respectively, had at least one comorbidity. COVID-19 clinical severity was moderate–severe in 77 and 89% of patients and critical in 8 and 4%, respectively. Supplemental oxygen was required by 47 and 55% of MM and noncancer patients, respectively, and 21%/9% vs 8%/6% required noninvasive/invasive ventilation. Inpatient mortality was 34 and 23% in MM and noncancer patients, respectively. Among MM patients, inpatient mortality was 41% in males, 42% in patients aged >65 years, 49% in patients with active/progressive MM at hospitalization, and 59% in patients with comorbid renal disease at hospitalization, which were independent prognostic factors on adjusted multivariate analysis. This case series demonstrates the increased risk and identifies predictors of inpatient mortality among MM patients hospitalized with COVID-19

EEG Authentication System Based on One- and Multi-Class Machine Learning Classifiers

19 páginas, 10 tablas, 3 figurasIn the current Information Age, it is usual to access our personal and professional information, such as bank account data or private documents, in a telematic manner. To ensure the privacy of this information, user authentication systems should be accurately developed. In this work, we focus on biometric authentication, as it depends on the user's inherent characteristics and, therefore, offers personalized authentication systems. Specifically, we propose an electrocardiogram (EEG)-based user authentication system by employing One-Class and Multi-Class Machine Learning classifiers. In this sense, the main novelty of this article is the introduction of Isolation Forest and Local Outlier Factor classifiers as new tools for user authentication and the investigation of their suitability with EEG data. Additionally, we identify the EEG channels and brainwaves with greater contribution to the authentication and compare them with the traditional dimensionality reduction techniques, Principal Component Analysis, and $\chi^2$ statistical test. In our final proposal, we elaborate on a hybrid system resistant to random forgery attacks \color{black} using an Isolation Forest and a Random Forest classifiers, obtaining a final accuracy of $82.3\%$, a precision of $91.1\%$ and a recall of $75.3\%$.This research was funded by the Spanish State Research Agency (AEI) of the Ministry of Sci- ence and Innovation (MCIN), project P2QProMeTe Grant No. PID2020-112586RBI00/AEI/10.13039/ 50110001103. This work was supported in part by Comunidad de Madrid (Spain) through Project CYNAMON, Grant No. P2018/TCS-4566-CM, co-funded by the European Regional Develop- ment Fund (ESF, FEDER and ERDF, EU); in part by the European Union’s Horizon 2020 Research and Innovation Program under Grant No. 872752 and under Grant No. 101017141; in part by the European Telecommunication Standard Institute (ETSI) technical committee (TC) on Smart Body Area Network (SmartBAN). L.H.-Á. would like to thank CSIC Project CASDiM for its support. E.B would like to thank UNICESV (Centro Universitario Di Ricerca Per Lo Sviluppo Competitivo Del Settore Vitivinicolo)

KeyEncoder: A secure and usable EEG-based cryptographic key generation mechanism

9 páginas, 5 figuras, 3 tablasNowadays, a rapid, easy, and convenient access to our private information is essential to carry out both personal and professional activities. In most cases, this information is sensitive and can be stolen due to its importance and the lack of security protocols. In this study we propose a time–invariant cryptographic key generation mechanism based on electroencephalogram (EEG) signals. We employed Discrete Wavelet Transform and autoencoders to extract the biometric features from the EEG signals. Using these features, we construct a scheme to generate secure seeds that can be used as inputs for secure hash functions and obtain cryptographic keys. The mechanism proposed preserves the privacy of the user, as the cryptographic key is generated for each new EEG signal received, avoiding the need of storing the key, previous EEG signals or any other information. Results show that the proposed mechanism is secure against random attacks, as a 0% of False Acceptance Rate is reported, while generating seeds with an entropy of 0.968 in less than 500 ms.This work was supported in part by the R&D&I project P2QProMeTe, Grant PID2020-112586RBI00 funded by MCIN/AEI/10.13039/ 501100011033; in part by the Madrid (Spain) Government under the multianual agreement with UC3M (“fostering young doctor research”, DEPROFAKE-CM-UC3M) and in the context of the V PRICIT research and technological innovation regional program; in part by the European Telecommunications Standards Institute (ETSI) SmartBAN; in part by the European Union’s Horizon 2020 Research and Innovation Pro- gramme under Grants 872752 and 101017331. L.H.A. thanks CSIC Project CASDiM for its supportPeer reviewe