Regulación de la función reparadora del ADN de la endonucleasa APE1 por nucleofosmina.

[ES] La proteína APE1 (endonucleasa apurínica(apirimidínica 1) es un factor clave en una de las rutas de reparación del ADN conocida como ruta de escisión de bases (BER), que se activa ante daños causados por estrés oxidativo, entre otros. APE1 reconoce sitios abásicos en el ADN, en los que la base nitrogenada se ha perdido por eliminación espontánea o por la acción de una glicosilasa de reparación, e hidroliza el enlace fosfodiéster en el lado 5´de dicho sitio abásico. Se ha descrito que la APE1 interacciona con la nucleofosmina (NPM1), una proteína abundante en el nucleolo con varias funciones que afectan a la homeostasis celular. NPM1 participa en la respuesta a estrés y la reparación del ADN, pero no se ha detallado aún su implicación en tales procesos, en particular, cómo podría colaborar con APE1 en la vía BER. En este trabajo se profundiza en el papel de la NPM1 en los mecanismos de reparación viendo cómo aumenta la actividad de APE1 sin llegarse a formar un complejo ternario APE1-NPM1-ADN. Este aumento podría estar relacionado con incrementar la afinidad de APE1 por ADN abásico por parte de NPM1, así como evitar la unión de APE1 a regiones no abásicas del ADN, donde no presenta función

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

Regulación de la función reparadora del ADN de la endonucleasa APE1 por nucleofosmina.

[ES] La proteína APE1 (endonucleasa apurínica(apirimidínica 1) es un factor clave en una de las rutas de reparación del ADN conocida como ruta de escisión de bases (BER), que se activa ante daños causados por estrés oxidativo, entre otros. APE1 reconoce sitios abásicos en el ADN, en los que la base nitrogenada se ha perdido por eliminación espontánea o por la acción de una glicosilasa de reparación, e hidroliza el enlace fosfodiéster en el lado 5´de dicho sitio abásico. Se ha descrito que la APE1 interacciona con la nucleofosmina (NPM1), una proteína abundante en el nucleolo con varias funciones que afectan a la homeostasis celular. NPM1 participa en la respuesta a estrés y la reparación del ADN, pero no se ha detallado aún su implicación en tales procesos, en particular, cómo podría colaborar con APE1 en la vía BER. En este trabajo se profundiza en el papel de la NPM1 en los mecanismos de reparación viendo cómo aumenta la actividad de APE1 sin llegarse a formar un complejo ternario APE1-NPM1-ADN. Este aumento podría estar relacionado con incrementar la afinidad de APE1 por ADN abásico por parte de NPM1, así como evitar la unión de APE1 a regiones no abásicas del ADN, donde no presenta función

Nucleophosmin interaction with APE1: Insights into DNA repair regulation

Nucleophosmin (NPM1), an abundant, nucleolar protein with multiple functions affecting cell homeostasis, has also been recently involved in DNA damage repair. The roles of NPM1 in different repair pathways remain however to be elucidated. NPM1 has been described to interact with APE1 (apurinic apyrimidinic endonuclease 1), a key enzyme of the base excision repair (BER) pathway, which could reflect a direct participation of NPM1 in this route. To gain insight into the possible role(s) of NPM1 in BER, we have explored the interplay between the subnuclear localization of both APE1 and NPM1, the in vitro interaction they establish, the effect of binding to abasic DNA on APE1 conformation, and the modulation by NPM1 of APE1 binding and catalysis on DNA. We have found that, upon oxidative damage, NPM1 is released from nucleoli and locates on patches throughout the chromatin, perhaps co-localizing with APE1, and that this traffic could be mediated by phosphorylation of NPM1 on T199. NPM1 and APE1 form a complex in vitro, involving, apart from the core domain, at least part of the linker region of NPM1, whereas the C-terminal domain is dispensable for binding, which explains that an AML leukemia-related NPM1 mutant with an unfolded C-terminal domain can bind APE1. APE1 interaction with abasic DNA stabilizes APE1 structure, as based on thermal unfolding. Moreover, our data suggest that NPM1, maybe by keeping APE1 in an “open” conformation, favours specific recognition of abasic sites on DNA, competing with off-target associations. Therefore, NPM1 might participate in BER favouring APE1 target selection as well as turnover from incised abasic DNA.This work was funded by the Spanish Ministry of Economy and FEDER (grant SAF2014-57743-R), the Basque Government (grant IT709-13) and the University of the Basque Country (grant GIU18/172)Peer reviewe

Evolución temporal en el tratamiento transcatéter de la estenosis aórtica: análisis del registro español de TAVI

Introduction and objectives: This study primary endpoint was to present the in-hospital all-cause mortality of the Spanish TAVI registry from its inception until 2018. Secondary endpoints included other in-hospital clinical events, 30-day all-cause mortality, and an assessment of the time trend of this registry. Methods: All consecutive patients included in the Spanish TAVI registry were analyzed. In this time-based analysis, the population was been divided into patients treated before 2014 (cohort A: 2009-2013) and patients treated between 2014 and 2018 (cohort B). Results: From August 2007 to June 2018, 7180 patients were included. The mean age was 81.2 ± 6.5 years and 53% were women. The logistic EuroSCORE was 12% (8-20). Transfemoral access was used in 89%. In-hospital and 30-day all-cause mortality was 4.7% and 5.7%, respectively. On the time-based analyses during the hospital stay, the rate of myocardial infarction, stroke, need for pacemakers, tamponade, coronary obstruction, and vascular complications was similar between both groups. However, cohort B showed less need for conversion to surgery and malapposition of the valve. Also, the implant success rate increased from 93% to 96% (P < .001). In-hospital and 30-day all-cause mortality was significantly lower in cohort B, ([OR, 0.65; IC95%, 0.48-0.86; P = .003] and [OR, 0.71; IC95%, 0.54-0.92; P = .002], respectively). Conclusions: The time trend analysis of the Spanish TAVI registry showed a change in the patients’ clinical profile and an improvement in the in-hospital clinical outcomes and 30-day all-cause mortality in patients treated more recently.Introducción y objetivos: El objetivo primario de este estudio fue presentar la mortalidad total intrahospitalaria del registro español de implante percutáneo de válvula aórtica (TAVI) desde su inicio hasta el año 2018, y como objetivos secundarios otros eventos clínicos intrahospitalarios, la mortalidad total a los 30 días y la evaluación de cuál ha sido la evolución temporal de este registro. Métodos: Fueron analizados todos los pacientes consecutivos incluidos en el registro español de TAVI. En este análisis temporal se dividió la población en pacientes tratados antes de 2014 (cohorte A: 2009-2013) y pacientes tratados entre los años 2014 y 2018 (cohorte B). Resultados: Desde agosto de 2007 hasta junio de 2018 se incluyeron 7.180 pacientes. La edad media fue de 81,2 ± 6,5 años y el 53% eran mujeres. El EuroSCORE logístico fue del 12% (8-20). Se utilizó un acceso transfemoral en el 89%. La mortalidad total intrahospitalaria fue del 4,7% y a los 30 días fue del 5,7%. En el análisis temporal durante la fase hospitalaria, las tasas de infarto, accidente cerebrovascular, necesidad de marcapasos, taponamiento, obstrucción coronaria y complicaciones vasculares fueron similares en ambos grupos. Sin embargo, en la cohorte B se observó una reducción de la necesidad de conversión a cirugía y de mala posición de la válvula, y además la tasa de éxito del implante fue mayor (93 frente a 96%; p < 0,001). La mortalidad por cualquier causa ajustada tanto intrahospitalaria como a los 30 días, fue significativamente menor en la cohorte B (odds ratio [OR] = 0,65; intervalo de confianza del 95% [IC95%], 0,48-0,86; p = 0,003; y OR = 0,71; IC95%, 0,54-0,92; p = 0,002, respectivamente). Conclusiones: En el análisis temporal del registro español de TAVI se observan un cambio en el perfil clínico de los pacientes y una mejora en la evolución clínica tanto intrahospitalaria como a los 30 días en los pacientes tratados en los últimos años

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field