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To investigate the effectiveness of a 3-year worksite lifestyle intervention on cardiovascular metrics and to study whether outcomes are influenced by baseline subclinical atherosclerosis (SA) by non-invasive imaging. A randomized controlled trial was performed to compare a lifestyle intervention with standard of care in asymptomatic middle-aged subjects, stratified by SA. The intervention consisted of nine motivational interviews during the first year, followed by three further sessions between Years 1 and 3. The primary outcome was the change in a pre-specified adaptation of the Fuster-BEWAT score (Blood pressure, Exercise, Weight, Alimentation, and Tobacco) between baseline and follow-up Years 1-3. A total of 1020 participants (mean age 50 ± 4 years) were enrolled, of whom 510 were randomly assigned to the intervention and 510 to the control group. The baseline adapted Fuster-BEWAT score was 16.2 ± 3.7 points in the intervention group and 16.5 ± 3.5 points in the control group. At Year 1, the score improved significantly in intervention participants compared with controls [estimate 0.83 (95% CI 0.52-1.15) points]. However, intervention effectiveness decreased to non-significant levels at Year 3 [0.24 (95% CI -0.10 to 0.59) points]. Over the 3-year period, the intervention was effective in participants having low baseline SA [0.61 (95% CI 0.30-0.93) points] but not in those with high baseline SA [0.19 (95% CI -0.26 to 0.64) points]. In middle-aged asymptomatic adults, a lifestyle intervention was associated with a significant improvement in cardiovascular health and behavioural metrics. The effect attenuated after 1 year as the intensity of the intervention was reduced. ClinicalTrials.gov (NCT02561065). TANSNIP-PESA is funded by Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC) Carlos III through an Investigator-initiated Study grant to Icahn School of Medicine from AstraZeneca. The PESA study is cofunded by the CNIC and Banco Santander. The study also received funding from the Instituto de Salud Carlos III (PI15/02019) and the European Regional Development Fund (ERDF) ‘A way to make Europe’. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033. The study funders were not involved in the study design; the collection, analysis, or interpretation of data; the writing of the report; or the decision to submit the paper for publication. Sí

Allergic diseases are allergen-induced immunological disorders characterized by the development of type 2 immunity and IgE responses. The prevalence of allergic diseases has been on the rise alike cardiovascular disease (CVD), which affects arteries of different organs such as the heart, the kidney and the brain. The underlying cause of CVD is often atherosclerosis, a disease distinguished by endothelial dysfunction, fibrofatty material accumulation in the intima of the artery wall, smooth muscle cell proliferation, and Th1 inflammation. The opposed T-cell identity of allergy and atherosclerosis implies an atheroprotective role for Th2 cells by counteracting Th1 responses. Yet, the clinical association between allergic disease and CVD argues against it. Within, we review different phases of allergic pathology, basic immunological mechanisms of atherosclerosis and the clinical association between allergic diseases (particularly asthma, atopic dermatitis, allergic rhinitis and food allergy) and CVD. Then, we discuss putative atherogenic mechanisms of type 2 immunity and allergic inflammation including acute allergic reactions (IgE, IgG1, mast cells, macrophages and allergic mediators such as vasoactive components, growth factors and those derived from the complement, contact and coagulation systems) and late phase inflammation (Th2 cells, eosinophils, type 2 innate-like lymphoid cells, alarmins, IL-4, IL-5, IL-9, IL-13 and IL-17). Severo Ochoa Center of Excellence, Grant/Award Number: CEX2020-001041- S; Pro CNIC Foundation; Ministerio de Ciencia e Innovación; Ministry of Science and Innovation, Grant/ Award Number: PID2019-110369RB- I00; European Commission, Grant/Award Number: ERC-CoG 819775 and H2020-HEALTH 945118; Spanish Ministry of Universities; Ayudas Margarita Salas para la Formación de Jóvenes Doctores—Universidad Autónoma de Madrid, Grant/ Award Number: CA1/RSUE/2021–00577; Formación de Profesorado Universitario, Grant/Award Number: FPU16/03953; Sociedad Española de Alergología e Inmunología Clínica (SEAIC), Grant/ Award Number: BECA20A9; New Frontiers in Research Fund, Grant/ Award Number: NFRFE-2019- 00083; The Nutricia Research Foundation, Grant/Award Number: NRF-2021- 13; Instituto de Salud Carlos III, Grant/Award Number: PI21/00158, PI21/01126, CP20/00043, PI18/01467, PI19/00044, RD16/0006/0015 and RD21/0002/0008; Severo Ochoa Program, Grant/Award Number: AEI/SEV-2017- 0712 Sí

Reciprocal interactions between endothelial cells (ECs) and adipocytes are fundamental to maintain white adipose tissue (WAT) homeostasis, as illustrated by the activation of angiogenesis upon WAT expansion, a process that is impaired in obesity. However, the molecular mechanisms underlying the crosstalk between ECs and adipocytes remain poorly understood. Here, we show that local production of polyamines in ECs stimulates adipocyte lipolysis and regulates WAT homeostasis in mice. We promote enhanced cell-autonomous angiogenesis by deleting Pten in the murine endothelium. Endothelial Pten loss leads to a WAT-selective phenotype, characterized by reduced body weight and adiposity in pathophysiological conditions. This phenotype stems from enhanced fatty acid β-oxidation in ECs concomitant with a paracrine lipolytic action on adipocytes, accounting for reduced adiposity. Combined analysis of murine models, isolated ECs and human specimens reveals that WAT lipolysis is mediated by mTORC1-dependent production of polyamines by ECs. Our results indicate that angiocrine metabolic signals are important for WAT homeostasis and organismal metabolism. We thank members of the Endothelial Pathobiology and Microenvironment Group for helpful discussions. We thank the CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. The research leading to these results has received funding from la Fundación BBVA (Ayuda Fundacion BBVA a Equipos de Investigación Científica 2019, PR19BIOMET0061) and from SAF2017-82072-ERC from Ministerio de Ciencia, Innovación y Universidades (MCIU) (Spain). The laboratory of M.G. is also supported by the research grants SAF2017-89116R-P (FEDER/EU) co-funded by European Regional Developmental Fund (ERDF), a Way to Build Europe and PID2020-116184RB-I00 from MCEI; by the Catalan Government through the project 2017-SGR; PTEN Research Foundation (BRR-17-001); La Caixa Foundation (HR19-00120 and HR21-00046); by la Asociación Española contra el Cancer-Grupos Traslacionales (GCTRA18006CARR, also to A.C.); European Foundation for the Study of Diabetes/Lilly research grant, also to M.C.); and by the People Programme (Marie Curie Actions; grant agreement 317250) of the European Union’s Seventh Framework Programme FP7/2007-2013 and the Marie Skłodowska-Curie (grant agreement 675392) of the European Union’s Horizon 2020 research. The laboratory of A.C. is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the department of education (IKERTALDE IT1106-16), the MCIU (PID2019-108787RB-I00 (FEDER/ EU); Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF2016-81975-REDT), La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17, the Vencer el Cancer foundation and the European Research Council (ERC) (consolidator grant 819242). CIBERONC was co-funded with FEDER funds and funded by Instituto de Salud Carlos III (ISCIII). The laboratory of M.C. is supported by the ERC under the European Union’s Horizon 2020 research and innovation programme (grant agreement 725004) and CERCA Programme/Generalitat de Catalunya (M.C.). The laboratory of D.S. is supported by research grants from MINECO (SAF2017- 83813-C3-1-R, also to L.H., cofounded by the ERDF), CIBEROBN (CB06/03/0001), Government of Catalonia (2017SGR278) and Fundació La Marató de TV3 (201627- 30). The laboratory of R.N. is supported by FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (RTI2018-099413-B-I00 and and RED2018-102379-T), Xunta de Galicia (2016-PG057 and 2020-PG015), ERC under the European Union’s Horizon 2020 research and innovation programme (grant agreement 810331), Fundación BBVA, Fundacion Atresmedia and CIBEROBN, which is an initiative of the ISCIII of Spain, which is supported by FEDER funds. The laboratory of J.A.V. is supported by research grants from MICINN (RTI2018-099250-B100) and by La Caixa Foundation (ID 100010434, LCF/PR/HR17/52150009). P.M.G.-R. is supported by ISCIII grant PI15/00701 cofinanced by the ERDF, A Way to Build Europe. Personal support was from Marie Curie ITN Actions (E.M.), Juan de la Cierva (IJCI-2015-23455, P.V.), CONICYT fellowship from Chile (S.Z.), Vetenskapsradet (Swedish Research Council, 2018-06591, L.G.) and NCI K99/R00 Pathway to Independence Award (K99CA245122, P. Castel). Sí

Aims Experimental studies suggest that increased bone marrow (BM) activity is involved in the association between cardiovascular risk factors and inflammation in atherosclerosis. However, human data to support this association are sparse. The purpose was to study the association between cardiovascular risk factors, BM activation, and subclinical atherosclerosis. Methods and results Whole body vascular 18F-fluorodeoxyglucose positron emission tomography/magnetic resonance imaging (18F-FDG PET/MRI) was performed in 745 apparently healthy individuals [median age 50.5 (46.8–53.6) years, 83.8% men] from the Progression of Early Subclinical Atherosclerosis (PESA) study. Bone marrow activation (defined as BM 18F-FDG uptake above the median maximal standardized uptake value) was assessed in the lumbar vertebrae (L3–L4). Systemic inflammation was indexed from circulating biomarkers. Early atherosclerosis was evaluated by arterial metabolic activity by 18F-FDG uptake in five vascular territories. Late atherosclerosis was evaluated by fully formed plaques on MRI. Subjects with BM activation were more frequently men (87.6 vs. 80.0%, P = 0.005) and more frequently had metabolic syndrome (MetS) (22.2 vs. 6.7%, P < 0.001). Bone marrow activation was significantly associated with all MetS components. Bone marrow activation was also associated with increased haematopoiesis—characterized by significantly elevated leucocyte (mainly neutrophil and monocytes) and erythrocyte counts—and with markers of systemic inflammation including high-sensitivity C-reactive protein, ferritin, fibrinogen, P-selectin, and vascular cell adhesion molecule-1. The associations between BM activation and MetS (and its components) and increased erythropoiesis were maintained in the subgroup of participants with no systemic inflammation. Bone marrow activation was significantly associated with high arterial metabolic activity (18F-FDG uptake). The co-occurrence of BM activation and arterial 18F-FDG uptake was associated with more advanced atherosclerosis (i.e. plaque presence and burden). Conclusion In apparently healthy individuals, BM 18F-FDG uptake is associated with MetS and its components, even in the absence of systemic inflammation, and with elevated counts of circulating leucocytes. Bone marrow activation is associated with early atherosclerosis, characterized by high arterial metabolic activity. Bone marrow activation appears to be an early phenomenon in atherosclerosis development. [Progression of Early Subclinical Atherosclerosis (PESA); NCT01410318].

Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice. Sí

18 p.-6 fig.-1 tab., Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS., Work supported by Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) (MCIN/AEI/https://doi.org/10.13039/501100011033 grants SAF2016-79490-R and PID2019-108489RB-I00), with co-funding from Fondo Social Europeo (“El FSE invierte en tu futuro”), and a donation from Asociación Progeria Alexandra Peraut. Microscopy was conducted at the Microscopy & Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/https://doi.org/10.13039/501100011033. R.M.N was supported by the Ministerio de Educación, Cultura y Deporte (pre-doctoral contract FPU16/05027), and I.B. is supported by the Comunidad Autónoma de Madrid (grants 2017-T1/BMD-5247 and 2021-5A/BMD-20944), and the Ramón y Cajal contract (RYC2021-033805-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union “NextGenerationEU”/PRTR. The CNIC is supported by the MCIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant number CEX2020-001041-S funded by MCIN/AEI/https://doi.org/10.13039/501100011033).

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism. Sí

Cellular senescence is a state of irreversible cell cycle arrest that often emerges after tissue damage and in age-related diseases. Through the production of a multicomponent secretory phenotype (SASP), senescent cells can impact the regeneration and function of tissues. However, the effects of senescent cells and their SASP are very heterogeneous and depend on the tissue environment and type as well as the duration of injury, the degree of persistence of senescent cells and the organism's age. While the transient presence of senescent cells is widely believed to be beneficial, recent data suggest that it is detrimental for tissue regeneration after acute damage. Furthermore, although senescent cell persistence is typically associated with the progression of age-related chronic degenerative diseases, it now appears to be also necessary for correct tissue function in the elderly. Here, we discuss what is currently known about the roles of senescent cells and their SASP in tissue regeneration in ageing and age-related diseases, highlighting their (negative and/or positive) contributions. We provide insight for future research, including the possibility of senolytic-based therapies and cellular reprogramming, with aims ranging from enhancing tissue repair to extending a healthy lifespan. Work in the authors’ laboratory is supported by MINECO-Spain (RTI2018-096068), H2020 European Research Council-2016-AdG-741966, LaCaixaHEALTH-HR17-00040, MWRF, French Muscular Dystrophy Association, Muscular Dystrophy Association, Fundacio LaMarató TV3 (80/19-202021 and 137/ 38-202033) and UPGRADE-H2020-825825; and María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370), Severo Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). Sí

V. Andrés’s laboratory is supported by Ministerio de Ciencia e Innovación (MICIN)/Agencia Estatal de Investigación (AEI) (grant PID2019-108489RBI00/AEI/10.13039/501100011033) with cofund from Fondo Social Europeo “El FSE invierte en tu futuro”. The CNIC is supported by the MICIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/ AEI/10.13039/501100011033). J. Díez is consultant to AstraZeneca, Bayer, and Vifor Pharma. Sí

Accurate detection of pathogenic single nucleotide variants (SNVs) is a key challenge in whole exome and whole genome sequencing studies. To date, several in silico tools have been developed to predict deleterious variants from this type of data. However, these tools have limited power to detect new pathogenic variants, especially in non-coding regions. In this study, we evaluate the use of a new metric, the Shannon Entropy of Locus Variability (SELV), calculated as the Shannon entropy of the variant frequencies reported in genome-wide population studies at a given locus, as a new predictor of potentially pathogenic variants in non-coding nuclear and mitochondrial DNA and also in coding regions with a selective pressure other than that imposed by the genetic code, e.g splice-sites. For benchmarking, SELV was compared to predictors of pathogenicity in different genomic contexts. In nuclear non-coding DNA, SELV outperformed CDTS (AUCSELV = 0.97 in ROC curve and PR-AUCSELV = 0.96 in Precision-recall curve). For non-coding mitochondrial variants (AUCSELV = 0.98 in ROC curve and PR-AUCSELV = 1.00 in Precision-recall curve) SELV outperformed HmtVar. Moreover, SELV was compared against two state-of-the-art ensemble predictors of pathogenicity in splice-sites, ada-score, and rf-score, matching their overall performance both in ROC (AUCSELV = 0.95) and Precision-recall curves (PR-AUC = 0.97), with the advantage that SELV can be easily calculated for every position in the genome, as opposite to ada-score and rf-score. Therefore, we suggest that the information about the observed genetic variability in a locus reported from large scale population studies could improve the prioritization of SNVs in splice-sites and in non-coding regions. FSC is supported by the Ministerio de Ciencia, Innovación, y Universidades (MCIU) [grant no. RTI2018-102084-B-I00]. JAE is supported by the MCIU (RTI2018-099357-BI00), Human Frontier Science Program (RGP0016/2018), CIBERFES16/10/00282 and RED2018-102576-T. The CNIC is supported by MCIU and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence [MCIU award SEV-XXX].” Sí

Instituto de Salud Carlos III (ISCIII), European Regional Development Fund/European Social Fund "A way to make Europe"/" Investing in your future" [PI18/0004, PI20/0320, PT17/0015/0043]; ISCIII; MCIN; Pro-CNIC Foundation; Severo Ochoa Centers of Excellence program [CEX2020-001041-S]; ISCIII [CM20/00101], Frutos F. de, Ochoa JP, Navarro-Peñalver M, Baas A, Bjerre JV, Zorio E, Méndez I, Lorca R, Verdonschot JAJ, García-Granja PE, Bilinska Z, Fatkin D, Fuentes-Cañamero ME, García-Pinilla JM, García-Álvarez MI, Girolami F, Barriales-Villa R, Díez-López C, Lopes LR, Wahbi K, García-Álvarez A, Rodríguez-Sánchez I, Rekondo-Olaetxea J, Rodríguez-Palomares JF, Gallego-Delgado M, Meder B, Kubanek M, Hansen FG, Restrepo-Córdoba MA, Palomino-Doza J, Ruiz-Guerrero L, Sarquella-Brugada G, Perez-Perez AJ, Bermúdez-Jiménez FJ, Ripoll-Vera T, Rasmussen TB, Jansen M, Sabater-Molina M, Elliot PM, Garcia-Pavia P; European Genetic Cardiomyopathies Initiative Investigators

This study was funded by the Spanish Ministry of Science and Innovation (“RETOS 2019” grant No PID2019-107332RB-I00 to B.I). B.I is funded by the European Commission (ERC-CoG grant No 819775, and H2020-HEALTH grant No 945118). J.N. is recipient of a predoctoral grant (Jordi Soler Soler) through CIBERCV. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministry of Science and Innovation and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). Sí

Protein-based hydrogels have been extensively studied in the field of biomaterials given their ability to mimic living tissues and their special resemblance to the extracellular matrix. Despite this, the methods used for the control of mechanical properties of hydrogels are very limited, focusing mainly on their elasticity, with an often unrealistic characterization of mechanical properties such as extensibility, stiffness and viscoelasticity. Being able to control these properties is essential for the development of new biomaterials, since it has been demonstrated that mechanical properties affect cell behaviour and biological processes. To better understand the mechanical behaviour of these biopolymers, a computational model is here developed to characterize the mechanical behaviour of two different protein-based hydrogels. Strain-stress tests and stress-relaxation tests are evaluated computationally and compared to the results obtained experimentally in a previous work. To achieve this goal the Finite Element Method is used, combining hyperelastic and viscoelastic models. Different hyperelastic constitutive models (Mooney-Rivlin, Neo-Hookean, first and third order Ogden, and Yeoh) are proposed to estimate the mechanical properties of the protein-based hydrogels by least-square fitting of the in-vitro uniaxial test results. Among these models, the first order Ogden model with a viscoelastic model defined in Prony parameters better reproduces the strain-stress response and the change of stiffness with strain observed in the in-vitro tests. APB was supported by MCIN/AEI/10.13039/501100011033/ and by European Union NextGeneration EU/PRT through the project PLEC2021-007709 (ProCanAid) the Aragon Institute for Engineering Research (I3A). SHR gratefully acknowledges the support of the Government of Aragon (Grant no 2019-23). The work of JMGA was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Advance grant agreement ICoMICS No 101018587) and the Spanish Ministry of Economy and Competitiveness, Spain Grant No PID2021-122409OBC21/AEI/10.13039/501100011033/ FEDER, UE. JAC acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN), Spain through grant BIO2017-83640-P (AEI/FEDER, UE). CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCIN, Spain and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence, Spain (grant CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011 033). CHL was the recipient of an FPI predoctoral fellowship, Spain (BES-2015-073191). Sí

This study was approved by the institutional ethical review board (CEIC) of Hospital 12 de Octubre (Madrid, Spain) and institutional review board information is04.625 version September 26th2018 on Act 20/18 dated by November 13th2018. Written informed consent was obtained from all participants. Conflict of Interest StatementJohan Skog has patents for exosome-based technologies and is an employee and shareholder of Bio-techne. J. Aquiles Sanchez is an employee of Bio-techne. Lisa Meyer, Daniel Enderle and Mikkel Noerholm are former employees of Bio-techne. Authors declare no additional conflicts of interes To the editor, cutaneous melanoma in its early stages is a curable disease by surgery of skin lesions. However, intermediate stages display variable outcomes. BRAF V600 mutations are the most frequent alterations in melanoma ocurring in 35-50% of patients ( Luke et al., 2017) and remain informative for guiding first-line treatment election ( Robert et al., 2019). We would like to thank Carmen María García, Sara Sánchez-Redondo, Vanesa Santos and Juan García-Agulló for technical support. This work has been funded by Fundación AECC (LABAE19027PEIN) (H.P.), Grupo Español de Melanoma (H.P.), Fundación BBVA (H.P.), FIS PI-17/00957 (P.L.O-R) and Centro de Excelencia “Severo Ochoa” (CEX2019-000891-S) (S.G-S.). No

The archetypical folded shape of the human cortex has been a long-standing topic for neuroscientific research. Nevertheless, the accurate neuroanatomical segmentation of sulci remains a challenge. Part of the problem is the uncertainty of where a sulcus transitions into a gyrus and vice versa. This problem can be avoided by focusing on sulcal fundi and gyral crowns, which represent the topological opposites of cortical folding. We present Automated Brain Lines Extraction (ABLE), a method based on Laplacian surface collapse to reliably segment sulcal fundi and gyral crown lines. ABLE is built to work on standard FreeSurfer outputs and eludes the delineation of anastomotic sulci while maintaining sulcal fundi lines that traverse the regions with the highest depth and curvature. First, it segments the cortex into gyral and sulcal surfaces; then, each surface is spatially filtered. A Laplacian-collapse-based algorithm is applied to obtain a thinned representation of the surfaces. This surface is then used for careful detection of the endpoints of the lines. Finally, sulcal fundi and gyral crown lines are obtained by eroding the surfaces while preserving the connectivity between the endpoints. The method is validated by comparing ABLE with three other sulcal extraction methods using the Human Connectome Project (HCP) test-retest database to assess the reproducibility of the different tools. The results confirm ABLE as a reliable method for obtaining sulcal lines with an accurate representation of the sulcal topology while ignoring anastomotic branches and the overestimation of the sulcal fundi lines. ABLE is publicly available via https://github.com/HGGM-LIM/ABLE . This work was supported by the project exAScale ProgramIng models for extreme Data procEssing (ASPIDE), that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 801091. This work has received funding from “la Caixa” Foundation under the project code LCF/PR/HR19/52160001. Susanna Carmona funded by Instituto de Salud Carlos III, co-funded by European Social Fund “Investing in your future” (Miguel Servet Type I research contract CP16/00096). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Yasser Alemán-Gómez is supported by the Swiss National Science Foundation (185897) and the National Center of Competence in Research (NCCR) SYNAPSY - The Synaptic Bases of Mental Diseases, funded as well by the Swiss National Science Foundation (51AU40-1257). Sí

Population aging and age-related cardiovascular disease (CVD) are becoming increasingly prevalent worldwide, generating a huge medical and socioeconomic burden. The complex regulation of aging and CVD and the interaction between these processes are crucially dependent on cellular stress responses. Interferon-stimulated gene-15 (ISG15) encodes a ubiquitin-like protein expressed in many vertebrate cell types that can be found both free and conjugated to lysine residues of target proteins via a post-translational process termed ISGylation. Deconjugation of ISG15 (deISGylation) is catalyzed by the ubiquitin-specific peptidase 18 (USP18). The ISG15 pathway has mostly been studied in the context of viral and bacterial infections and in cancer. This minireview summarizes current knowledge on the role of ISG15 in age-related telomere shortening, genomic instability, and DNA damage accumulation, as well as in hypertension, diabetes, and obesity, major CVD risk factors prevalent in the elderly population. VA lab is supported by Ministerio de Ciencia e Innovación (MICINN)/Agencia Estatal de Investigación (AEI) (grant PID2019- 108489RB-I00/AEI/10.13039/501100011033) with cofund from Fondo Social Europeo “El FSE invierte en tu futuro”, the Progeria Research Foundation (grant PRF2019–77), Asociación Progeria Alexandra Peraut, Fundació Marató TV3 (grant 202033- 31), and Sociedad Española de Cardiología (SEC/FEC-INV-BAS 22/ 005). MG-A is supported by the MCINN (post-doctoral contract FJC2021-047576-I). The CNIC is supported by the MICINN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033) Sí

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli. We thank R. Parton (Institute for Molecular Biosciences, Queensland), P. Pilch (Boston University School of Medicine) and L. Liu (Boston University School of Medicine) for kindly providing PTRFKO cells and reagents, S. Casas Tintó for kindly providing SH-Sy5y cells, P. Bassereau (Curie Institute, Paris) for kindly providing OT setup, V. Labrador Cantarero from CNIC microscopy Unit for helping with ImageJ analysis, O. Otto and M. Herbig for providing help with RTDC experiments, S. Berr and K. Gluth for technical assistance in cell culture, F. Steiniger for support in electron tomography, and A. Norczyk Simón for providing pCMV-FLAG-PTRF construct. This project received funding from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 641639; grants from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033): SAF2014-51876-R, SAF2017-83130-R co-funded by ‘ERDF A way of making Europe’, PID2020-118658RB-I00, PDC2021-121572-100 co-funded by ‘European Union NextGenerationEU/PRTR’, CSD2009- 0016 and BFU2016-81912-REDC; and the Asociación Española Contra el Cáncer foundation (PROYE20089DELP) all to M.A.d.P. M.A.d.P. is member of the Tec4Bio consortium (ref. S2018/NMT¬4443; Comunidad Autónoma de Madrid/FEDER, Spain), co-recipient with P.R.-C. of grants from Fundació La Marató de TV3 (674/C/2013 and 201936- 30-31), and coordinator of a Health Research consortium grant from Fundación Obra Social La Caixa (AtheroConvergence, HR20-00075). M.S.-A. is recipient of a Ramón y Cajal research contract from MCIN (RYC2020-029690-I). The CNIC Unit of Microscopy and Dynamic Imaging is supported by FEDER ‘Una manera de hacer Europa’ (ReDIB ICTS infrastructure TRIMA@CNIC, MCIN). We acknowledge the support from Deutsche Forschungsgemeinschaft through grants to M.M.K. (KE685/7-1) and B.Q. (QU116/6-2 and QU116/9-1). Work in D.N. laboratory was supported by grants from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 812772 and MCIN (DPI2017-83721-P). Work in C.L. laboratory was supported by grants from Curie, INSERM, CNRS, Agence Nationale de la Recherche (ANR-17-CE13-0020-01) and Fondation ARC pour la Recherche (PGA1-RF20170205456). Work in P.R.-C. lab is funded by the MCIN (PID2019-110298GB-I00), the EC (H20 20-FETPROACT-01-2016-731957). Work in X.T. lab is funded by the MICIN (PID2021-128635NB-I00), ERC (Adv-883739) and La Caixa Foundation (LCF/PR/HR20/52400004; co-recipient with P.R.-C.). IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

The study was partially funded by the ‘Accion Transversal del Cancer’, approved by the Spanish Ministry Council on 11 October 2007, by the Instituto de Salud Carlos III-FEDER (PI08/1770, PI08/1359, PI09/00773, PI09/01286, PI09/01903, PI09/02078, PI09/01662, PI11/01403, PI11/01889, PI11/02213; PI12/00265, PI12/01270, PI12/00715, PI12/00150, PI14/01219, PI14/0613, PI15/00069, PI15/00914, PI15/01032, PI17CIII/00034, CIBERESP CB06/02/0073), the Fundación Marqués de Valdecilla (API 10/09), the Junta de Castilla y León (LE22A10- 2), the Consejería de Salud of the Junta de Andalucía (PI-0571-2009, PI-0306-2011, salud201200057018tra), the Conselleria de Sanitat of the Generalitat Valenciana (AP_061/10), the Recercaixa (2010ACUP 00310), the Regional Government of the Basque Country, the European Commission grants FOOD-CT-2006-036224-HIWATE, the Spanish Association Against Cancer (AECC) Scientific Foundation, the Catalan Government DURSI grant 2014SGR647, 2017SGR723, 2017SGR1085 and 2014SGR850, the Fundación Caja de Ahorros de Asturias and by the University of Oviedo. MCT is funded by a Ramón y Cajal fellowship (RYC-2017-01892) from the Spanish Ministry of Science, Innovation and Universities and co-funded by the European Social Fund. ISGlobal acknowledges support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program., Supplementary Information The online version contains supplementary material available at https://doi.org/10.1038/s41598-022-25789-9., Breast and prostate cancers have been associated with circadian disruption. Some previous studies examined associations of sleep duration and breast or prostate cancer risk though findings remain inconsistent. This study examines associations of a range of detailed sleep characteristics and breast and prostate cancer risk in a large-scale population-based case–control study, MCC-Spain. A total of 1738 incident breast cancer cases, 1112 prostate cancer cases and frequency matched controls (n = 1910, and 1493 respectively) were recruited. Detailed data on habitual sleep duration, quality, timing, and daytime napping (“siesta”) were collected at recruitment. Additional data on sleep habits during both the previous year and at age 40 years were also subsequently captured. Adjusted odds ratios (ORs) and 95% confidence intervals (CI) were estimated. There were no associations of habitual sleep duration (h), timing of sleep, or any or specific sleep problems, and either breast and prostate cancer risk. There was a significant positive association of ever taking habitual siestas at recruitment and breast cancer risk (OR = 1.22, 95% CI 1.06–1.42), which strengthened with increased frequency or duration. There were also significant positive associations observed for both breast and prostate cancer, among those reporting recent sleep problems, but not sleep problems at age 40 years, in a subsequent circadian questionnaire. Adverse associations with siesta and disturbed sleep during the previous year likely reflect symptoms of developing/diagnosed cancer and comorbidities. Overall, there was no clear association between various sleep characteristics and breast or prostate cancer risk observed., Basque Country, Catalan Government DURSI 2014SGR647, 2014SGR850, 2017SGR1085, 2017SGR723, Consejería de Salud of the Junta de Andalucía PI-0306-2011, PI-0571-2009, Spanish Association Against Cancer, Fundación Bancaria Caja de Ahorros de Asturias, Ministerio de Ciencia, Innovación y Universidades, Eno Scientific Foundation, European Commission FOOD-CT-2006-036224-HIWATE, Fundación Científica Asociación Española Contra el Cáncer, Generalitat de Catalunya, Instituto de Salud Carlos III CIBERESP CB06/02/0073, PI08/1359, PI08/1770, PI09/00773, PI09/01286, PI09/01662, PI09/01903, PI09/02078, PI11/01403, PI11/01889, PI11/02213, PI12/00150, PI12/00265, PI12/00715, PI12/01270, PI14/01219, PI14/0613, PI15/00069, PI15/00914, PI15/01032, PI17CIII/00034, Ministerio de Ciencia e Innovación CEX2018-000806-S, European Social Fund, Universidad de Oviedo RYC-2017-01892, Consejería de Educación, Junta de Castilla y León LE22A10-2, Conselleria de Sanitat Universal i Salut Pública 2010ACUP 00310, AP_061/10, Fundación Marqués de Valdecilla API 10/09

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multiple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-specific virally delivered Cre expression to delete either p110α or p110β (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost exclusively supported by p110α activity, whereas p110β controls neurotransmitter release and metabotropic glutamate receptor-dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110β jointly contribute to N-methyl-d-aspartate receptor-dependent postsynaptic long-term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110β. This work was supported by the Spanish Ministry of Science and Innovation grants SAF2017-86983-R and PID2020-117651RB (to J.A.E.), Spanish Ministry of Science and Innovation grants SAF2017-89116R-P (FEDER/EU) and PID2020-116184RB (to M.G.), Carlos III Institute of Health-Fondo de Investigación Sanitaria grant PRB3 (IPT17/0019–ISCIII-SGEFI/ERDF, ProteoRed) and CIBERCV (to J.A.L.), Spanish Ministry of Economy postdoctoral contract IJCI-2015-25507 (to M.I.C.), Marie Curie cofund UAM-UE (EU project 713366) Intertalentum Postdoctoral Program (to V.B.), and Spanish Ministry of Science and Innovation predoctoral contracts (to C.S.-C., A.F.-R., and S.L.-G.). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

The integrated analysis of different omic layers can provide new knowledge not provided by their individual analysis. This approach is also necessary to validate data and reveal post-transcriptional and post-translational mechanisms of gene expression regulation. In this work, we validated the possibility of applying this approach to non-model species such as Quercus ilex. Transcriptomics, proteomics, and metabolomics from Q. ilex seedlings subjected to drought-like conditions under the typical summer conditions in southern Spain were integrated using a non-targeted approach. Two integrative approaches, PCA and DIABLO, were used and compared. Both approaches seek to reduce dimensionality, preserving the maximum information. DIABLO also allows one to infer interconnections between the different omic layers. For easy visualization and analysis, these interconnections were analyzed using functional and statistical networks. We were able to validate results obtained by analyzing the omic layers separately. We identified the importance of protein homeostasis with numerous protease and chaperones in the networks. We also discovered new key processes, such as transcriptional control, and identified the key function of transcription factors, such as DREB2A, WRKY65, and CONSTANS, in the early response to drought. This research was funded by the project ENCINOMICS-2 PID2019-109038RB-I00 of the Spanish Ministry of Economy and Competitiveness. V.M.G.-S. was supported by the Youth Employment Operational Program (EJI-17-AGR-164), co-financed by the Regional Government of Andalusia and the European Social Fund (ESF). M.-D.R., M.Á.C. and M.E. are grateful for being awarded the Juan de la Cierva-Incorporación (IJC2018-035272-I), Ramon y Cajal (RYC-2017-23706), and Juan de la Cierva-Formación (FJCI-2017-31613) contracts, respectively, by the Spanish Ministry of Science, Innovation and Universities. C.L.-H. is grateful to the Regional Government of Asturias, Spain, for being awarded the Severo Ochoa contract BP17-112. Sí

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Here, we show that dual deficiency for hematopoietic RXRa and RXRb induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRa and RXRb maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRa;RXRb-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRa;RXRb-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging. We thank the members of the J.A.C. and M.R. laboratories for extensive discussions and critiques of the manuscript. We thank Daniel Metzger (Université de Strasbourg, France) for Rxrbf/f 418 mice, Juan Carlos Zúñiga-Pflücker (Sunnybrook Health Sciences Centre, Canada) for OP9-NL1 cells, Daniel Jiménez-Carretero (CNIC) for t-SNE analysis, the CRG (Barcelona, Spain) Genomics Unit for ATACseq sequencing, and S. Bartlett (CNIC) for editorial assistance. We also thank the staff of the CNIC Cellomics and Animal facilities for technical support. This study was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICIN) (SAF2017-90604-REDT-NurCaMein, RTI2018- 095928-B100, and PID2021-122552OB-I00), La Marató de TV3 Foundation (201605-32), and the Comunidad de Madrid (MOIR-B2017/BMD-3684) to M.R and from the Formación de Profesorado Universitario (FPU17/01731) program (MICIN) to J.P. The project also received funding from the US National Institutes of Health (R01 DK124115, P01 HL158688, R01 HL147536, R01 CA237016 and U54 DK126108 to J.A.C). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

Telomere length (TL) is associated with biological aging, consequently influencing the risk of age-related diseases such as Alzheimer’s disease (AD). We aimed to evaluate the potential causal role of TL in AD endophenotypes (i.e., cognitive performance, N = 2233; brain age and AD-related signatures, N = 1134; and cerebrospinal fluid biomarkers (CSF) of AD and neurodegeneration, N = 304) through a Mendelian randomization (MR) analysis. Our analysis was conducted in the context of the ALFA (ALzheimer and FAmilies) study, a population of cognitively healthy individuals at risk of AD. A total of 20 single nucleotide polymorphisms associated with TL were used to determine the effect of TL on AD endophenotypes. Analyses were adjusted by age, sex, and years of education. Stratified analyses by APOE-ɛ4 status and polygenic risk score of AD were conducted. MR analysis revealed significant associations between genetically predicted longer TL and lower levels of CSF Aβ and higher levels of CSF NfL only in APOE-ɛ4 non-carriers. Moreover, inheriting longer TL was associated with greater cortical thickness in age and AD-related brain signatures and lower levels of CSF p-tau among individuals at a high genetic predisposition to AD. Further observational analyses are warranted to better understand these associations., The project leading to these results has received funding from the Alzheimer’s Association (Grant AARG-19-618265). This project has received funding from Instituto de Salud Carlos III (PI19/00119). Additional support has been received from “la Caixa” Foundation (ID 100010434), under agreement LCF/PR/GN17/50300004, the Alzheimer’s Association and an international anonymous charity foundation through the TriBEKa Imaging Platform project (TriBEKa-17-519007), the Health Department of the Catalan Government (Health Research and Innovation Strategic Plan (PERIS) 2016-2020 grant# SLT002/16/00201), and the Universities and Research Secretariat, Ministry of Business and Knowledge of the Catalan Government under the grant no. 2017-SGR-892. All CRG authors acknowledge the support of the Spanish Ministry of Science, Innovation and Universities to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. NV-T is funded by a post-doctoral grant, Juan de la Cierva Incorporación Programme (IJC2020-043216-I), Ministry of Science and Innovation–Spanish State Research Agency. EMAU is supported by the Spanish Ministry of Science, Innovation and Universities - Spanish State Research Agency (RYC2018-026053-I). MS-C receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement no. 948677). MS-C also receives funding from the Instituto de Salud Carlos III (PI19/00155) and from the Spanish Ministry of Science, Innovation and Universities (Juan de la Cierva Programme grant IJC2018-037478-I). HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532); the European Research Council (#681712); Swedish State Support for Clinical Research (#ALFGBG-720931); the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862); the AD Strategic Fund and the Alzheimer’s Association (#ADSF-21-831376-C, #ADSF-21-831381-C, and #ADSF-21-831377-C); the Olav Thon Foundation; the Erling-Persson Family Foundation, Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden (#FO2019-0228); the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 860197 (MIRIADE); and the UK Dementia Research Institute at UCL. KB is supported by the Swedish Research Council (#2017-00915); the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615); the Swedish Alzheimer Foundation (#AF-742881); Hjärnfonden, Sweden (#FO2017-0243); the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement (#ALFGBG-715986); the European Union Joint Program for Neurodegenerative Disorders (JPND2019-466-236); and the National Institute of Health (NIH), USA, (grant #1R01AG068398-01). JDG is supported by the Spanish Ministry of Science and Innovation (RYC-2013-13054). AS-V. is the recipient of an Instituto de Salud Carlos III Miguel Servet II fellowship (CP II 17/00029).

The p.Glu109Lys variant (Glu89Lys) is a rare cause of hereditary transthyretin amyloidosis (ATTRv) for which clinical spectrum remains unresolved. We sought to describe the clinical characteristics and outcomes of ATTR Glu89Lys amyloidosis and assess a potential founder effect in Spain. Patients with the p.Glu109Lys ATTRv variant from 14 families were recruited at 7 centres. Demographics, complementary tests and clinical course were analysed. Haplotype analysis was performed in 7 unrelated individuals. Thirty-eight individuals (13 probands, mean age 40.4 ± 13.1 years) were studied. After median follow-up of 5.1 years (IQR 1.7-9.6), 7 patients died and 7 required heart transplantation (median age at transplantation 50.5 years). Onset of cardiac and neurological manifestations occurred at a mean age of 48.4 and 46.8 years, respectively. Median survival from birth was 61.6 years and no individual survived beyond 65 years. Patients treated with disease-modifying therapies exhibited better prognosis (p

The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers. This study was supported by grants PID2019-104776RB-I00 and PID2020-120326RB-I00, CB16/11/00399 (CIBER CV) financed by MCIN/AEI/10.13039/501100011033, a grant from the Fundación BBVA (Ref. BIO14_298), and a grant from Fundació La Marató de TV3 (Ref. 20153431) to J.L.d.l.P. M.S.-A. was supported by a PhD contract from the Severo Ochoa Predoctor-al Program (SVP-2014-068723) of the MCIN/AEI/10.13039/501100011033. J.R.G.-B. was supported by SEC/FEC-INV-BAS 21/021. A.R. was funded by grants from MCIN (PID2021123925OB-I00), TerCel (RD16/0011/0024), AGAUR (2017-SGR-899), and Fundació La Marató de TV3 (201534-30). J.M.P.-P. was supported by RTI2018-095410-B-I00 (MCIN) and PY2000443 (Junta de Andalucía). B.I. was supported by the European Commission (H2020-HEALTH grant No. 945118) and by MCIN (PID2019-107332RB-I00). DO’R was sup-ported by the Medical Research Council (MC-A658-5QEB0) and KAMcG by the British Heart Foundation (RG/19/6/34387, RE/18/4/34215). The cost of this publication was supported in part with funds from the European Regional Devel-opment Fund. The Centro Nacional de Investigaciones Cardiovasculares is sup-ported by the ISCIII, the MCIN, and the Pro Centro Nacional de Investigaciones Cardiovasculares Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020001041-S) financed by MCIN/AEI/10.13039/501100011033. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising. Sí

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active β1-integrin content on the surface of Cav1KO fibroblasts. Florescence recovery after photobleaching analysis and endocytosis/recycling assays revealed that active β1-integrin is mostly endocytosed through the clathrin independent carrier/glycosylphosphatidyl inositol (GPI)-enriched endocytic compartment pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven β1-integrin activation is lower in Cav1KO mouse embryonic fibroblasts (MEFs) than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.Cells can physically sense their immediate environment by pulling and pushing through integrins, a type of proteins which connects the inside and outside of a cell by being studded through the cellular membrane. This sensing role can only be performed when integrins are in an active state. Two main mechanisms regulate the relative amount of active integrins: one controls the activation of the proteins already at the cell surface; the other, known as recycling, impacts how many new integrins are delivered to the membrane. Both processes are affected by changes in cell membrane tension, which is itself controlled by dimples (or ‘caveolae’ – little caves in Latin) present in the cell surface. Caveolae limit acute changes in tension by taking in (pinching off the dimples) or releasing (dimples flattening) segments of the membrane. However, it is still unclear how integrins and caveolae mechanically interact to regulate the ability for a cell to read its environment. To understand this process, Lolo et al. focused on mouse cells genetically manipulated to not build caveolae on their surfaces, and which cannot properly sense mechanical changes in their surroundings. These were exposed to beads covered in an integrin-binding protein and manipulated using magnetic tweezers. The manipulation showed that mutated cells bound to the beads more strongly than non-modified cells, indicating that they had more active integrins on their surface. This change was due to both an accelerated recycling mechanism (which resulted in more integrin being brought at the surface) and an increase in integrin activation (which was triggered by a higher membrane tension). Caveolae therefore couple mechanical inputs to integrin recycling and activation. Healthy tissues rely on cells correctly sensing physical changes in their environment so they can mount an appropriate response. This ability, for example, is altered in cancerous cells which start to form tumours. The findings by Lolo et al. bring together physics and biology to provide new insights into the potential mechanisms causing such impairments.

Atherosclerosis is a chronic inflammatory disease of the arteries that can lead to thrombosis, infarction, and stroke and is the leading cause of mortality worldwide. Immunization of pro-atherogenic mice with malondialdehyde-modified low-density lipoprotein (MDA-LDL) neo-antigen is athero-protective. However, the immune response to MDA-LDL and the mechanisms responsible for this athero-protection are not completely understood. Here, we find that immunization of mice with MDA-LDL elicits memory B cells, plasma cells, and switched anti-MDA-LDL antibodies as well as clonal expansion and affinity maturation, indicating that MDA-LDL triggers a bona fide germinal center antibody response. Further, Prdm1fl/flAicda-Cre+/kiLdlr-/- pro-atherogenic chimeras, which lack germinal center-derived plasma cells, show accelerated atherosclerosis. Finally, we show that MDA-LDL immunization is not athero-protective in mice lacking germinal-center-derived plasma cells. Our findings give further support to the development of MDA-LDL-based vaccines for the prevention or treatment of atherosclerosis. We thank all members of the B Cell Biology Laboratory for useful discussions, A. Rodriguez-Ronchel for the design of the graphical abstract, V.G. de Ye´benes for critical reading of the manuscript, and V. Labrador for help with microscopy and image analysis. I.M.-F. and C.L. were fellows of the research training program funded by Ministerio de Economı´a y Competitividad (SVP-2014-068216 and SVP-2014-068289, respectively), A.d.M.M. is funded by ‘‘la Caixa’’ Foundation HR17–00247, and A.R.R. was supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC). The project leading to these results has received funding from la Caixa Banking Foundation under the project code HR17-00247 and from SAF2016-75511-R and PID2019-106773RB-I00/AEI/10.13039/501100 011033 grants to A.R.R. (Plan Estatal de Investigacio´ n Cientı´fica y Te´ cnica y de Innovacio´ n 2013–2016 Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigacio´ n: Proyectos I + D + i 2016, Ministerio de Economı´a,Industria y Competitividad) and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). CIBERCV (J.L.M.-V.) and CIBERDEM (J.C.E.-G.) are Instituto de Salud Carlos III projects. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacio´ n (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa institute (CEX2020-001041-S grant funded by MCIN/AEI/10.13039/501100011033). Sí

Acute myocarditis is an inflammatory condition that may herald the onset of dilated cardiomyopathy (DCM) or arrhythmogenic cardiomyopathy (ACM). We investigated the frequency and clinical consequences of DCM and ACM genetic variants in a population-based cohort of patients with acute myocarditis. This was a population-based cohort of 336 consecutive patients with acute myocarditis enrolled in London and Maastricht. All participants underwent targeted DNA sequencing for well-characterized cardiomyopathy-associated genes with comparison to healthy controls (n=1053) sequenced on the same platform. Case ascertainment in England was assessed against national hospital admission data. The primary outcome was all-cause mortality. Variants that would be considered pathogenic if found in a patient with DCM or ACM were identified in 8% of myocarditis cases compared with

Neural stem cells (NSCs) are self-renewing, multipotent cells which give rise to all components of the central nervous system (CNS) during embryogenesis, but also activate in response to injury and disease and maintain a certain level of neurogenic activity throughout adulthood. This activity takes place in specialized regions of the brain, the neurovascular niches, whose main role is to control the behaviour of the CNS. In adult mammals, two main "canonical" niches have been described: The subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus. This review discusses our current understanding of the neural stem cells and their canonical niches, as well as their structure, behaviours, and role in neural disease. This research was funded by the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III grants PI18/00462 to M.V.G.-G. and PT20/00044 to M.D., was cofinanced by the European Regional Development Fund, “A way of making Europe” initiative. This work was partially supported by Comunidad de Madrid (S2017/BMD-3867 RENIM-CM) and cofinanced by the European Structural and Investment Fund. This study was also supported by the Plan Estatal de I+D+I 2013–2016, with funding from the European Regional Development Fund (ERDF), “A way to build Europe” initiative. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation and is a Severo Ochoa Centre of Excellence (SEV-2015-0505). Sí

To study the relationship between urinary total polyphenol excretion (TPE) in adolescents and ideal cardiovascular (CVH) metrics. 1151 adolescents aged 12.04 (0.46) years participating in the SI! Program for Secondary Schools were selected based on the availability of urine samples and information required to assess CVH metrics. Data on health behaviours (smoking status, body mass index, physical activity, and healthy diet) and health factors (blood pressure, total cholesterol, and blood glucose) were used to calculate the CVH metrics. TPE in urine was analysed by a Folin-Ciocalteu method after solid-phase extraction. Associations between TPE (categorized into tertiles) and CVH metrics (total and separate scores) were assessed using multilevel mixed-effect regression models. Higher TPE levels were associated with higher (healthier) CVH scores and ideal smoking status (OR 1.54, 95% CI 1.10; 1.87, p value = 0.007), physical activity (OR 1.12, 95% CI 1.02; 1.23, p value = 0.022) and total cholesterol (OR 1.78, 95% CI 1.16; 2.73, p value = 0.009) after multivariate adjustment. An association between TPE and total CVH scores was observed only in boys. Girls with higher TPE had higher rates of ideal total cholesterol and blood pressure. According to our findings, higher urinary TPE is related to better CVH scores, with relevant differences in this association by gender. The authors wish to thank all the volunteers and their families, teachers, and schools for their contribution to the SI! Program for Secondary Schools. The SI! Program for Secondary Schools trial was supported by the SHE Foundation, the “la Caixa” Foundation (grant numbers LCF/PR/CE16//10700001 and LCF/PR/MS19/12220001), and the Fundació la Marató de TV3 (grant number 369/C/2016). Support was also provided by the Ministerio de Ciencia, Innovación y Universidades (grant number AGL2016–75329-R), and Generalitat de Catalunya. Rodrigo Fernández-Jiménez is a recipient of grant PI19/01704 funded by the Fondo de Investigación Sanitaria- Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund/European Social Fund "A way to make Europe"/"Investi ng in your future". The Centro Nacional de Investigaciones Cardiovasculares is supported by the ISCIII, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). Anna Tresserra-Rimbau was supported by a Serra Húnter Fellowship. Emily P. Laveriano-Santos is thankful for the FI-SDUR (EMC/3345/2020) fellowship from the Generalitat de Catalunya. Isabella Parilli-Moser thanks the FI-SDUR (EMC/2703/2019) fellowship. Sí

The holm oak (Quercus ilex L.) is the dominant tree species of the Mediterranean forest and the Spanish agrosilvopastoral ecosystem, "dehesa." It has been, since the prehistoric period, an important part of the Iberian population from a social, cultural, and religious point of view, providing an ample variety of goods and services, and forming the basis of the economy in rural areas. Currently, there is renewed interest in its use for dietary diversification and sustainable food production. It is part of cultural richness, both economically (tangible) and environmentally (intangible), and must be preserved for future generations. However, a worrisome degradation of the species and associated ecosystems is occurring, observed in an increase in tree decline and mortality, which requires urgent action. Breeding programs based on the selection of elite genotypes by molecular markers is the only plausible biotechnological approach. To this end, the authors' group started, in 2004, a research line aimed at characterizing the molecular biology of Q. ilex. It has been a challenging task due to its biological characteristics (long life cycle, allogamous, high phenotypic variability) and recalcitrant nature. The biology of this species has been characterized following the central dogma of molecular biology using the omics cascade. Molecular responses to biotic and abiotic stresses, as well as seed maturation and germination, are the two main objectives of our research. The contributions of the group to the knowledge of the species at the level of DNA-based markers, genomics, epigenomics, transcriptomics, proteomics, and metabolomics are discussed here. Moreover, data are compared with those reported for Quercus spp. All omics data generated, and the genome of Q. ilex available, will be integrated with morphological and physiological data in the systems biology direction. Thus, we will propose possible molecular markers related to resilient and productive genotypes to be used in reforestation programs. In addition, possible markers related to the nutritional value of acorn and derivate products, as well as bioactive compounds (peptides and phenolics) and allergens, will be suggested. Subsequently, the selected molecular markers will be validated by both genome-wide association and functional genomic analyses. This research was supported by the grant ENCINOMICS-2 PID2019-109038RB-I00/AEI/ 10.13039/501100011033 from the Spanish Ministry of Economy and Competitiveness. C.L.-H. is grateful for the contract “Severo Ochoa Program (BP17-112, Government of Principado de Asturias, Spain)”. M.Á.C. and M.-D.R. are grateful for the awards of Ramón y Cajal (RYC-2017-23706), and the Juan de la Cierva-Incorporación (IJC2018-035272-I) contracts from the Spanish Ministry of Science, Innovation and Universities, respectively. Sí

As evidenced by scintigraphy imaging, the prevalence of transthyretin (TTR) cardiac amyloidosis in heart failure patients with preserved ejection fraction (HFpEF) and left ventricular hypertrophy (LVH) ranges between 13% and 19%. The natural evolution of cardiac amyloidosis begins with the deposition of amyloid material in the myocardium, with LVH ensuing at later stages. With current imaging modalities, it is possible to detect TTR cardiac amyloidosis before the hypertrophic stage. The aim of this study was to determine the prevalence of TTR cardiac amyloidosis in HFpEF patients without LVH. The study prospectively enrolled patients admitted for HF with LV ejection fraction (LVEF) ≥ 50% and LV wall thickness

Background Even when low-density lipoprotein-cholesterol (LDL-C) levels are lower than guideline thresholds, a residual risk of atherosclerosis remains. It is unknown whether triglyceride (TG) levels are associated with subclinical atherosclerosis and vascular inflammation regardless of LDL-C. Objectives This study sought to assess the association between serum TG levels and early atherosclerosis and vascular inflammation in apparently healthy individuals. Methods An observational, longitudinal, and prospective cohort study, including 3,754 middle-aged individuals with low to moderate cardiovascular risk from the PESA (Progression of Early Subclinical Atherosclerosis) study who were consecutively recruited between June 2010 and February 2014, was conducted. Peripheral atherosclerotic plaques were assessed by 2-dimensional vascular ultrasound, and coronary artery calcification (CAC) was assessed by noncontrast computed tomography, whereas vascular inflammation was assessed by fluorine-18 fluorodeoxyglucose uptake on positron emission tomography. Results Atherosclerotic plaques and CAC were observed in 58.0% and 16.8% of participants, respectively, whereas vascular inflammation was evident in 46.7% of evaluated participants. After multivariate adjustment, TG levels ≥150 mg/dl showed an association with subclinical noncoronary atherosclerosis (odds ratio [OR]: 1.35; 95% confidence interval [CI]: 1.08 to 1.68; p = 0.008). This association was significant for groups with high LDL-C (OR: 1.42; 95% CI: 1.11 to 1.80; p = 0.005) and normal LDL-C (OR: 1.85; 95% CI: 1.08 to 3.18; p = 0.008). No association was found between TG level and CAC score. TG levels ≥150 mg/dl were significantly associated with the presence of arterial inflammation (OR: 2.09; 95% CI: 1.29 to 3.40; p = 0.003). Conclusions In individuals with low to moderate cardiovascular risk, hypertriglyceridemia was associated with subclinical atherosclerosis and vascular inflammation, even in participants with normal LDL-C levels. (Progression of Early Subclinical Atherosclerosis [PESA]; NCT01410318), Central Illustration

Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development. This work was supported by grants BFU2017-84914-P and PID2020-115755GB-I00 from the Spanish State Research Agency (AEI). M.P. was supported by fellowships from the Comunidad de Madrid (PEJD-2018-PRE/BMD-9079) and the Ministerio de Ciencia e Innovación (PRE2018-083477). The CBMSO is supported by an Institutional grant from the Fundación Ramon Areces, and the the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence grant (CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Deposited in PMC for immediate release. Sí

Circadian nutritional behaviors, defined by the daily eating/fasting cycle, have been linked with breast cancer. This study aimed to further disentangle the association of nighttime fasting duration and time of breakfast with breast cancer risk. We analyzed data from 1,181 breast cancer cases and 1,326 population controls from the Spanish multicase-control study (MCC-Spain), 2008–2013. We collected circadian nutritional behaviors at mid-age via a telephonic interview. We applied logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CIs) for the association of nighttime fasting duration and time of breakfast with breast cancer risk in all women and stratified by menopausal status. Models were adjusted for age, center, education, family history of breast cancer, age at menarche, number of children, breastfeeding, age at first child, body mass index (BMI), contraceptive use, and hormonal replacement therapy (HRT). A later time of breakfast was associated with a non-significant increased risk of breast cancer (OR = 1.05, 95% CI: 0.95–1.16, per hour increase). This association was stronger among premenopausal women, among whom each hour later, the time of breakfast was associated with an 18% increase in breast cancer risk (OR = 1.18, 95% CI: 1.01–1.40). The association was not observed in postmenopausal women. We did not observe an association between nighttime fasting duration and breast cancer risk after adjusting for the time of breakfast. In this study, late breakfast was associated with increased breast cancer risk, especially among premenopausal women, compared with early breakfast. Aside from nutritional quality, circadian nutritional behaviors should be further studied in relation to cancer., This study was partially funded by the “Accion Transversal del Cancer,” approved on the Spanish Ministry Council on the 11 October 2007, Instituto de Salud Carlos III-FEDER (PI08/1770, PI08/0533, PI08/1359, PS09/00773, PS09/01286, PS09/01903, PS09/02078, PS09/01662, PI11/01889, PI11/02213, PI12/00488, PI12/01270, PI12/00715, PI14/01219, PI14/0613, and PI17/01388), Fundación Marqués de Valdecilla (API 10/09), the ICGC International Cancer Genome Consortium CLL [The ICGC CLL-Genome Project was funded by Spanish Ministerio de Economía y Competitividad (MINECO) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) del ISCIII (RD12/0036/0036)], the Junta de Castilla y León (LE22A10- 2), the Consejería de Salud of the Junta de Andalucía (PI-0571-2009, PI-0306-2011, and salud201200057018tra), the Conselleria de Sanitat of the Generalitat Valenciana (AP_061/10), the Recercaixa (2010ACUP 00310), the Regional Government of the Basque Country, the Consejería de Sanidad de la Región de Murcia, by the European Commission grants FOOD-CT-2006-036224-HIWATE, the Spanish Association Against Cancer (AECC) Scientific Foundation, by the Catalan Government—Agency for Management of University and Research Grants (AGAUR) grants 2017SGR723 and 2014SGR850, the Fundación Caja de Ahorros de Asturias, and the University of Oviedo. ISGlobal acknowledges support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S) and support from the Generalitat de Catalunya through the CERCA Program. AP-C was supported by the MINECO (Ministry of Economy in Spain) Grant no. PRE2019-089038, fellowship.

Aims: Although systemic embolism is a potential complication in transthyretin amyloid cardiomyopathy (ATTR-CM), data about its incidence and prevalence are scarce. We studied the incidence, prevalence and factors associated with embolic events in ATTR-CM. Additionally, we evaluated embolic events according to the type of oral anticoagulation (OAC) and the performance of the CHA2DS2-VASc score in this setting. Methods and results: Clinical characteristics, history of atrial fibrillation (AF) and embolic events were retrospectively collected from ATTR-CM patients evaluated at four international amyloid centres. Overall, 1191 ATTR-CM patients (87% men, median age 77.1 years [interquartile range-IQR 71.4–82], 83% ATTRwt) were studied. A total of 162 (13.6%) have had an embolic event before initial evaluation. Over a median follow-up of 19.9 months (IQR 9.9–35.5), 41 additional patients (3.44%) had an embolic event. Incidence rate (per 100 patient-years) was 0 among patients in sinus rhythm with OAC, 1.3 in sinus rhythm without OAC, 1.7 in AF with OAC, and 4.8 in AF without OAC. CHA2DS2-VASc did not predict embolic events in patients in sinus rhythm whereas in patients with AF without OAC, only those with a score ≥4 had embolic events. There was no difference in the incidence rate of embolism between patients with AF treated with vitamin K antagonists (VKAs) (n = 322) and those treated with direct oral anticoagulants (DOACs) (n = 239) (p = 0.66). Conclusions: Embolic events were a frequent complication in ATTR-CM. OAC reduced the risk of systemic embolism. Embolic rates did not differ with VKAs and DOACs. The CHA2DS2-VASc score did not correlate well with clinical outcome in ATTR-CM and should not be used to assess thromboembolic risk in this population, This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects “PI18/0765 & PI20/01379” (Co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”). The CNIC is supported by the ISCIII, MCIN, the Pro-CNIC Foundation, and the Severo Ochoa Centers of Excellence program (CEX2020-001041-S)

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools. This manuscript was funded by grants PDI-2020-120412RB-I00 and PDC2021- 121719-I00 (FS-M) and PID2020- 119352RB-I00 (AS) from the Spanish Ministry of Economy and Competitiveness; CAM (S2017/BMD3671-INFLAMUNE-CM) from the Comunidad de Madrid (FS-M). CIBERCV (CB16/11/00272) and BIOIMID PIE13/041 from the Instituto de Salud Carlos. The current research has received funding from 'la Caixa' Foundation under the project code HR17-00016. Grants from Ramón Areces Foundation 'Ciencias de la Vida y de la Salud' (XIX Concurso-2018) and from Ayuda Fundación BBVA y Equipo de Investigación Científica (BIOMEDICINA-2018) (to FSM). The CNIC is supported by the Ministerio de Ciencia, Innovacion y Universidades and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015–0505). IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (MINECO, CEX2020-001039-S). SGD is supported by a grant from the Spanish Ministry of Universities. Authors thank Dr Miguel Vicente-Manzanares for critical review and editing. We also thank Dr Francisco Urbano and Dr Covadonga Aguado for their support with EM (TEM facilities, Universidad Autónoma de Madrid). Sí

Andersen Tawil Syndrome (ATS) is a rare inheritable disease associated with loss-of-function mutations in KCNJ2, the gene coding the strong inward rectifier potassium channel Kir2.1, which forms an essential membrane protein controlling cardiac excitability. ATS is usually marked by a triad of periodic paralysis, life-threatening cardiac arrhythmias and dysmorphic features, but its expression is variable and not all patients with a phenotype linked to ATS have a known genetic alteration. The mechanisms underlying this arrhythmogenic syndrome are poorly understood. Knowing such mechanisms would be essential to distinguish ATS from other channelopathies with overlapping phenotypes and to develop individualized therapies. For example, the recently suggested role of Kir2.1 as a countercurrent to sarcoplasmic calcium reuptake might explain the arrhythmogenic mechanisms of ATS and its overlap with catecholaminergic polymorphic ventricular tachycardia (CPVT). Here we summarize current knowledge on the mechanisms of arrhythmias leading to sudden cardiac death in ATS. We first provide an overview of the syndrome and its pathophysiology, from the patient´s bedside to the protein, and discuss the role of essential regulators and interactors that could play a role in cases of ATS. The review highlights novel ideas related to some post-translational channel interactions with partner proteins that might help define the molecular bases of the arrhythmia phenotype. We then propose a new all-embracing classification of the currently known ATS loss-of-function mutations according to their position in the Kir2.1 channel structure and their functional implications. We also discuss specific ATS pathogenic variants, their clinical manifestations and treatment stratification. The goal is to provide a deeper mechanistic understanding of the syndrome toward the development of novel targets and personalized treatment strategies. This work has been supported by SQTS Project (PI20/01220) of the public call Proyectos de investigación en salud 2020 (PI-FIS-2020) funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, (FEDER/FSE), ‘Una manera de hacer Europa’/’El FSE invierte en tu futuro’; Fundación ‘La Caixa’: Macromoleculopathies (LCF/PR/ HR19/52160013) of the call HEALTH RESEARCH 2018; Genotype Specific Arrhythmogenic Mechanisms in Andersen-Tawil Syndrome (736/C/2020) of the call Ayudas a la investigación en enfermedades raras 2020 (LAMARATO-2020)’ amb el suport de la Fundació La Marató de TV3; the European Union’s Horizon 2020 research and innovation programme under grant agreement (GA-965286) (‘Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.’); the Dynamic Microscopy and Imaging Unit - ICTS-ReDib Grant ICTS-2018-04-CNIC-16 funded by MCIN/AEI/10.13039/501100011033 and ERDF ‘A way of making Europe’; EQC2018-005070-P funded by MCIN/AEI/10.13039/ 501100011033 and FEDER ‘Una manera de hacer Europa’; AIM-M holds a FPU contract (FPU20/01569) from Ministerio de Universidades; LKG holds a FPI contract (PRE2018-083530), Ministerio de Economía y Competitividad de España co-funded by Fondo Social Europeo ‘El Fondo Social Europeo invierte en tu futuro’, attached to the Project SEV-2015-0505-18-2; MLVP holded a contract (PEJD-2019-PRE/ BMD-15982) funded by Consejería de Educación e Investigación de la Comunidad de Madrid y Fondo Social Europeo ‘El FSE invierte en tu futuro’; IM-C holds a PFIS contract (FI21/00243) funded by Instituto de Salud Carlos III and Fondo Social Europeo Plus (FSE+), ‘Cofunded by the European Union’; The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Centre of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/ 10.13039/501100011033). Sí

There is evidence that in schizophrenia, imbalances in inflammatory and oxidative processes occur during pregnancy and in the early postnatal period, generating interest in the potential therapeutic efficacy of anti-inflammatory and antioxidant compounds. Mangiferin is a polyphenolic compound abundant in the leaves of Mangifera indica L. that has robust antioxidant and anti-inflammatory properties, making it a potential candidate for preventive or co-adjuvant therapy in schizophrenia. Hence, this study set-out to evaluate the effect of mango leaf extract (MLE) in a model of schizophrenia based on maternal immune activation, in which Poly I:C (4 mg/kg) is administered intravenously to pregnant rats. Young adult (postnatal day 60-70) or adolescent (postnatal day 35-49) male offspring received MLE (50 mg/kg of mangiferin) daily, and the effects of MLE in adolescence were compared to those of risperidone, assessing behavior, brain magnetic resonance imaging (MRI), and oxidative/inflammatory and antioxidant mediators in the adult offspring. MLE treatment in adulthood reversed the deficit in prepulse inhibition (PPI) but it failed to attenuate the sensitivity to amphetamine and the deficit in novel object recognition (NOR) induced. By contrast, adolescent MLE treatment prevented the sensorimotor gating deficit in the PPI test, producing an effect similar to that of risperidone. This MLE treatment also produced a reduction in grooming behavior, but it had no effect on anxiety or novel object recognition memory. MRI studies revealed that adolescent MLE administration partially counteracted the cortical shrinkage, and cerebellum and ventricle enlargement. In addition, MLE administration in adolescence reduced iNOS mediated inflammatory activation and it promoted the expression of biomarkers of compensatory antioxidant activity in the prefrontal cortex and hippocampus, as witnessed through the reduction of Keap1 and the accumulation of NRF2 and HO1. Together, these findings suggest that MLE might be an alternative therapeutic or preventive add-on strategy to improve the clinical expression of schizophrenia in adulthood, while also modifying the time course of this disease at earlier stages in populations at high-risk. EB, JAG-P and ST-S work was supported by the “Fondo Europeo de Desarrollo Regional” (FEDER)-UE “A way to build Europe” from the “Ministerio de Economía y Competitividad” (RTI2018-099778-B-I00); from the “Plan Nacional sobre Drogas, Ministerio de Sanidad, Consumo y Bienestar Social” (2019I041); from the “Ministerio de Salud-Instituto de Salud Carlos III” (PI18/01691); from the “Programa Operativo de Andalucía FEDER, Iniciativa Territorial Integrada ITI 2014-2020 Consejería Salud y Familias, Junta de Andalucía” (PI-0080- 2017, PI-0009-2017), “Consejería de Salud y Familias, Junta de Andalucía” (PI-0134-2018 and PEMP-0008-2020); from the “Consejería de Transformación Económica, Industria, Conocimiento y Universidad, Junta de Andalucía” (P20_00958 and CTS-510); from the CEIMAR (CEIJ-003); from the “Instituto de Investigación e Innovación en Ciencias Biomédicas de Cádiz-INiBICA” (LI19/06IN-CO22; IN-C09); from the “CIBERSAM”: CIBER-Consorcio Centro de Investigación Biomédica en Red- (CB07/09/0033), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 955684. CM, LC and MTF-P were supported by the Spanish Ministry of Science and Technology (PID2020- 116229RB-I00) and European Regional Development Fund (ERDF). KM and JCL were supported by the “MICINN” (PID2019-109033RB-I00) and the “CIBERSAM”: CIBERConsorcio Centro de Investigación Biomédica en Red- (CB07/ 09/0026), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. MLS-M was supported by the “Ministerio de Ciencia, Innovación, Instituto de Salud Carlos III” (PI17/ 01766, BA21/00030), co-financed by European Regional Development Fund (ERDF), “A way to make Europe”; from the “CIBERSAM”: CIBERConsorcio Centro de Investigación Biomédica en Red- (CB07/09/0031), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación; from the “Delegación del Gobierno para el Plan Nacional sobre Drogas” (2017/085); from the “Fundación Mapfre” and “Fundación Alicia Koplowitz.” MD work was supported by the “Ministerio de Ciencia e In review 18/ 28 Innovación” (MCIN) and “Instituto de Salud Carlos III” (ISCIII) (PT20/00044); from the “CIBERSAM” CIBERConsorcio Centro de Investigación Biomédica en Red-(CB07/ 09/0031). The CNIC is supported by the ISCIII, the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs., This work was funded by the Spanish government (grants BFU2017-84914-P and PID2020-115755GB-I00 to M.M.; BFU2016-74961-P and BFU2016-81887-REDT to J.L.G.-S.), the Andalusian government (grant BIO-396 to J.L.G.-S.), and the European Research Council (ERC; grant agreement 740041 to J.L.G.-S.). M.T. held Juan de la Cierva fellowships from the Spanish government (FJCI-2017-31791 and IJC2019-038897-I), R.R. and R.D.A. held FPU fellowships from the government, and J.V. was the recipient of a “La Caixa” fellowship. Work in the laboratory of J.L.G.-S. was supported by a María de Maetzu Unit of Excellence Grant (MDM-2016-0687) to the Department of Gene Regulation and Morphogenesis of the CABD. The CBMSO is supported by an institutional grant from the Fundación Ramon Areces, and the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). : With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020-001041-S).

The variation on birth weight is associated with several outcomes early on in life and low birth weight (LBW) increases the risk of morbidity and mortality. Some environmental exposures during pregnancy, such as particulate matters and other traffic-related pollutants can have a significant effect on pregnant women and fetuses. The aim of this study is to estimate the effect of exposure to traffic density during pregnancy over birth weight in Spain, from 2000-2017. This was a retrospective, cross-sectional study using the information from Spain Birth Registry Statistics database. The traffic density was measured using the Annual average daily traffic. Multivariate linear regression models using birth weight and traffic density were performed, as well as a logistic regression model to estimated Odds ratios for LBW and GAM models to evaluate the non-linear effect. Our findings showed that increases in traffic density were associated with reduction of birth weight and increases of LBW risk. Moreover, exposure to high and very-high traffic-density during pregnancy were associated with reduction of birth weight and increase on LBW risk comparing with exposure to low number of cars trespassing the neighborhoods. The results of this study agree with previous literature and highlights the need of effective policies for reducing traffic density in residential neighborhoods of cities and towns. This work was supported by the Spanish Consortium for Research on Epidemiology and Public Health(CIBERESP) (ESP20PI01/2020) and Spanish Institute of Health Carlos III (PI19CIII-00025. Mònica Guxens is funded by a Miguel Servet II fellowship (CPII18/00018) awarded by the Spanish Institute of Health Carlos III. We also acknowledge support from the Spanish Ministry of Science and Innovation and the State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. Sí

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), whose outbreak in 2019 led to an ongoing pandemic with devastating consequences for the global economy and human health. According to the World Health Organization, COVID-19 has affected more than 481 million people worldwide, with 6 million confirmed deaths. The joint efforts of the scientific community have undoubtedly increased the pace of production of COVID-19 vaccines, but there is still so much uncharted ground to cover regarding the mechanisms of SARS-CoV-2 infection, replication and host response. These issues can be approached by proteomics with unprecedented capacity paving the way for the development of more efficient strategies for patient care. In this study, we present a deep proteome analysis that has been performed on a cohort of 72 COVID-19 patients aiming to identify serum proteins assessing the dynamics of the disease at different age ranges. A panel of 53 proteins that participate in several functions such as acute-phase response and inflammation, blood coagulation, cell adhesion, complement cascade, endocytosis, immune response, oxidative stress and tissue injury, have been correlated with patient severity, suggesting a molecular basis for their clinical stratification. Eighteen protein candidates were further validated by targeted proteomics in an independent cohort of 84 patients including a group of individuals that had satisfactorily resolved SARS-CoV-2 infection. Remarkably, all protein alterations were normalized 100 days after leaving the hospital, which further supports the reliability of the selected proteins as hallmarks of COVID-19 progression and grading. The optimized protein panel may prove its value for optimal severity assessment as well as in the follow up of COVID-19 patients., This study was supported by competitive grants from the Spanish Ministry of Science, Innovation and Universities (PGC2018-097019-B-I00), the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria grant PRB3 (PT17/0019/0003-ISCIII-SGEFI/ERDF, ProteoRed), and “la Caixa” Banking Foundation (project code HR17-00247). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). The CNB was supported by the Carlos III Health Institute of Spain -Fondos FEDER (EU) (PRB3-ISCIII, supported by grant PT17/0019/0001). Comunidad de Madrid Grant B2017/BMD-3817. Severo Ochoa Project SEV 2017-0712. Intramural CSIC PIE/COVID-19 projects 202020E079 and 202020E108. This research work was also funded by the European Commission—NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global).

Cutaneous melanoma is one of the most aggressive and deadliest cancers in human beings due to its invasiveness and other factors. Histopathological analysis is crucial for a proper diagnosis. Optical tissue clearing is a novel field that allows 3D image acquisition of large-scale biological tissues. Optical clearing and immunolabeling for 3D fluorescence imaging has yet to be extensively applied to melanoma. In the present manuscript, we establish, for the first time, an optical clearing and immunostaining procedure for human melanoma and human cell line-derived melanoma xenograft models using the CUBIC (clear, unobstructed brain imaging cocktails) technique. We have successfully cleared the samples and achieved 3D volumetric visualization of the tumor microenvironment, vasculature, and cell populations. This study has been funded by Instituto de Salud Carlos III through the projects “PI18/00462” and “PT20/00044”, co-funded by the European Union (ERDF, “A way to make Europe”). This work was partially supported by Comunidad de Madrid (S2017/BMD-3867 RENIM-CM), and co-financed by European Structural and Investment Fund. The CNIC is supported by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Publicado

Life on Earth anticipates recurring 24-hour environmental cycles via genetically encoded molecular clocks active in all mammalian organs. Communication between these clocks controls circadian homeostasis. Intertissue communication is mediated, in part, by temporal coordination of metabolism. Here, we characterize the extent to which clocks in different organs control systemic metabolic rhythms, an area that remains largely unexplored. We analyzed the metabolome of serum from mice with tissue-specific expression of the clock gene Bmal1. Having functional hepatic and muscle clocks can only drive a minority (13%) of systemic metabolic rhythms. Conversely, limiting Bmal1 expression to the central pacemaker in the brain restores rhythms to 57% of circulatory metabolites. Rhythmic feeding imposed on clockless mice resulted in a similar rescue, indicating that the central clock mainly regulates metabolic rhythms via behavior. These findings explicate the circadian communication between tissues and highlight the importance of the central clock in governing those signals. P.P. was funded by the Wenner-Gren Foundation; the Foundation Blanceflor Boncompagni Ludovisi, née Bildt; and the Tore Nilsson Foundation for Medical Science. Funding for P.S.-C. was provided by the National Institutes of Health (NIH) (AG053592 and DK114652), a Novo Nordisk Foundation Challenge Grant, and Institut National de la Sante et la Recherche Medicale (U1233 INSERM, France). Research in the S.A.B. laboratory is supported partially by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 787041), the Government of Cataluña (SGR grant), the Government of Spain (MINECO), the La Marató/TV3 Foundation, the Foundation Lilliane Bettencourt, the Spanish Association for Cancer Research (AECC), and the Worldwide Cancer Research Foundation (WCRF). The IRB Barcelona is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). P.M.C. acknowledges funding from MICINN-RTI2018-096068, ERC-2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM, DPP-Spain, Fundació La Marató TV3-80/19-202021, MWRF, María-de-Maeztu Program for Units of Excellence to UPF (MDM-2014-0370), and the Severo- Ochoa Program for Centers of Excellence to CNIC (SEV-2015-0505). T.S. was supported by a Japan Society for the Promotion of Science (JSPS) fellowship. C.M.G. was funded by European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 749869. T.M. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 754510. P.-S.W. is supported by grant RYC2019-026661-I funded by MCIN/ AEI/10.13039/501100011033 and by “ESF Investing in your future.” We acknowledge predoctoral fellowships to M.C. from the NIH (GM117942), the American Heart Association (17PRE33410952), and the UCI School of Medicine Behrens Research Excellence Award. The work of S.C. and P.B. was, in part, supported by NIH grant NIH GM123558. Sí

Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients. To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays. Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers. We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias. Supported by National Institutes of Health R01 HL122352 grant; 'la Caixa' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB. Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB. Sí

This study aimed to develop and validate a liquid chromatography/electrospray ionization-linear ion trap quadrupole-Orbitrap-high-resolution mass spectrometry (HPLC/ESI-LTQ-Orbitrap-HRMS) method to identify and quantify urinary microbial phenolic metabolites (MPM), as well as to explore the relationship between MPM and dietary (poly)phenols in Spanish adolescents. A total of 601 spot urine samples of adolescents aged 12.02 ± 0.41 years were analyzed. The quantitative method was validated for linearity, limit of detection, limit of quantification, recovery, intra- and inter-day accuracy and precision, as well as postpreparative stability according to the criteria established by the Association of Official Agricultural Chemists International. A total of 17 aglycones and 37 phase II MPM were identified and quantified in 601 spot urine samples. Phenolic acids were the most abundant urinary MPM, whereas stilbenes, hydroxytyrosol, and enterodiol were the least abundant. Urinary hydroxycoumarin acids (urolithins) were positively correlated with flavonoid and total (poly)phenol intake. An HPLC-ESI-LTQ-Orbitrap-HRMS method was developed and fully validated to quantify MPM. The new method was performed accurately and is suitable for MPM quantification in large epidemiological studies. Urinary lignans and urolithins are proposed as potential biomarkers of grain and nut intake in an adolescent population. This research was funded in part by CICYT (AGL2016-75329-R and PID2020-114022RBI00]), the Instituto de Salud Carlos III—ISCIII (CIBEROBN CB12/03/30020) from the Ministry of Science, Innovation, and Universities (AEI/FEDER, UE) and Generalitat de Catalunya (GC) 2017 SGR 196. The SI! Program for Secondary School trial was funded by the Fundació la Marató de TV3 (369/C/2016), the SHE Foundation-‘la Caixa’ Foundation (LCF/PR/CE16/10700001), the Ministerio de Ciencia e Innovación (AGL2016-75329-R). E.P.L.-S. is supported by the FI-SDUR (EMC/3345/2020) fellowship from the Generalitat de Catalunya. M.M.-M. and S.C.-B. are supported by the Ministry of Science, Innovation, and Universities for the FPU17/00513 and FPU17/00785 fellowship. A.V.-Q. is supported the Ministry of Science, Innovation, and Universities for the Ramon y Cajal contract (RYC-2016-19355). A.T.-R. is supported by Serra Húnter Fellowship JM-G is a postgraduate fellow of the Ministerio de Ciencia e Innovación of Spain at the Residencia de Estudiantes (2020–ongoing). G.S.-B. is supported by a “la Caixa” Foundation fellowship (LCF/PR/MS19/12220001). R.F.-J. is a recipient of grant PI19/01704 funded by the Fondo de Investigación Sanitaria- Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”. The Centro Nacional de Investigaciones Cardiovasculares is supported by the ISCIII, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). Sí

Carotid and femoral plaque burden is a recognized biomarker of cardiovascular disease risk. A new electronic-sweep 3-dimensional (3D)-matrix transducer method can improve the functionality and image quality of vascular ultrasound atherosclerosis imaging. This study aimed to validate this method for plaque volume measurement in early and intermediate-advanced plaques in the carotid and femoral territories. Plaque volumes were measured ex vivo in pig carotid and femoral artery specimens by 3-dimensional vascular ultrasound (3DVUS) using a 3D-matrix (electronic-sweep) transducer and its associated 3D plaque quantification software, and were compared with gold-standard histology. To test the clinical feasibility and accuracy of the 3D-matrix transducer, an experiment was conducted in intermediate-high risk individuals with carotid and femoral atherosclerosis. The results were compared with those obtained using the previously validated mechanical-sweep 3D transducer and established 2-dimensional (2D)-based plaque quantification software. In the ex vivo study, the authors assessed 19 atherosclerotic plaques (plaque volume, 0.76 µL-56.30 μL), finding strong agreement between measurements with the 3D-matrix transducer and the histological gold-standard (intraclass correlation coefficient [ICC]: 0.992; [95% CI: 0.978-0.997]). In the clinical analysis of 20 patients (mean age 74.6 ± 4.45 years; 40% men), the authors found 64 (36 carotid and 28 femoral) of 80 scanned territories with atherosclerosis (measured atherosclerotic volume, 10 μL-859 μL). There was strong agreement between measurements made from electronic-sweep and mechanical-sweep 3DVUS transducers (ICC: 0.997 [95% CI: 0.995-0.998]). Agreement was also high between plaque volumes estimated by the 2D and 3D plaque quantification software applications (ICC: 0.999 [95% CI: 0.998-0.999]). Analysis time was significantly shorter with the 3D plaque quantification software than with the 2D multislice approach with a mean time reduction of 46%. 3DVUS using new matrix transducer technology, together with improved 3D plaque quantification software, simplifies the accurate volume measurement of early (small) and intermediate-advanced plaques located in carotid and femoral arteries. This study was partially funded by grants from the Ministerio de Economia, Industria y Competividad (MEIC) with cofunding from the European Regional Development Fund (ERDF) (SAF2016-75580-R to Dr Bentzon) and (BES-2016-076633 to Dr Nogales). Research funding was also received from the Instituto de Salud Carlos III Spain (PIE16/ 00021 to Drs Bueno and Fuster). The CNIC is supported by the Min- isterio de Ciencia, Innovacion y Universidades (MICINN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). This study forms part of a Master Research Agreement between the CNIC and Philips Healthcare. Drs Sánchez- González, Entrekin, and Collet-Billon are employees of Philips Healthcare. All other authors have reported that they have no re- lationships to disclose related to the contents of this paper. Sí

Aims: Genotype and left ventricular scar on cardiac magnetic resonance (CMR) are increasingly recognized as risk markers for adverse outcomes in non-ischaemic dilated cardiomyopathy (DCM). We investigated the combined influence of genotype and late gadolinium enhancement (LGE) in assessing prognosis in a large cohort of patients with DCM. Methods and results: Outcomes of 600 patients with DCM (53.3 ± 14.1 years, 66% male) who underwent clinical CMR and genetic testing were retrospectively analysed. The primary endpoints were end-stage heart failure (ESHF) and malignant ventricular arrhythmias (MVA). During a median follow-up of 2.7 years (interquartile range 1.3–4.9), 24 (4.00%) and 48 (8.00%) patients had ESHF and MVA, respectively. In total, 242 (40.3%) patients had pathogenic/likely pathogenic variants (positive genotype) and 151 (25.2%) had LGE. In survival analysis, positive LGE was associated with MVA and ESHF (both, p < 0.001) while positive genotype was associated with ESHF (p = 0.034) but not with MVA (p = 0.102). Classification of patients according to genotype (G+/G−) and LGE presence (L+/L−) revealed progressively increasing events across L−/G−, L−/G+, L+/G− and L+/G+ groups and resulted in optimized MVA and ESHF prediction (p < 0.001 and p = 0.001, respectively). Hazard ratios for MVA and ESHF in patients with either L+ or G+ compared with those with L−/G− were 4.71 (95% confidence interval: 2.11–10.50, p < 0.001) and 7.92 (95% confidence interval: 1.86–33.78, p < 0.001), respectively. Conclusion: Classification of patients with DCM according to genotype and LGE improves MVA and ESHF prediction. Scar assessment with CMR and genotyping should be considered to select patients for primary prevention implantable cardioverter-defibrillator placement, This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) (PI18/0004, PI19/01283, PI20/0320). (Co-funded by European Regional Development Fund/European Social Fund ‘A way to make Europe’/‘Investing in your future’). The Hospital Universitario Puerta de Hierro Majadahonda, the Hospital Clinic, the Hospital Vall d’Hebron, the Hospital General Universitario Gregorio Marañón and the Hospital Universitario Virgen de la Arrixaca are members of the European Reference Network for rare, low-prevalence, and complex diseases of the heart (ERN GUARD-Heart). The CNIC is supported by the ISCIII, MCIN, the Pro-CNIC Foundation, and the Severo Ochoa Centers of Excellence program (CEX2020-001041-S). Conflict of interest: none declared

Clonal hematopoiesis of indeterminate potential (CHIP), defined as the presence of somatic mutations in cancer-related genes in blood cells in the absence of hematological cancer, has recently emerged as an important risk factor for several age-related conditions, especially cardiovascular disease. CHIP is strongly associated with normal aging, but its role in premature aging syndromes is unknown. Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare genetic condition driven by the accumulation of a truncated form of the lamin A protein called progerin. HGPS patients exhibit several features of accelerated aging and typically die from cardiovascular complications in their early teens. Previous studies have shown normal hematological parameters in HGPS patients, except for elevated platelets, and low levels of lamin A expression in hematopoietic cells relative to other cell types in solid tissues, but the prevalence of CHIP in HGPS remains unexplored. To investigate the potential role of CHIP in HGPS, we performed high-sensitivity targeted sequencing of CHIP-related genes in blood DNA samples from a cohort of 47 HGPS patients. As a control, the same sequencing strategy was applied to blood DNA samples from middle-aged and elderly individuals, expected to exhibit a biological age and cardiovascular risk profile similar to HGPS patients. We found that CHIP is not prevalent in HGPS patients, in marked contrast to our observations in individuals who age normally. Thus, our study unveils a major difference between HGPS and normal aging and provides conclusive evidence that CHIP is not frequent in HGPS and, therefore, is unlikely to contribute to the pathophysiology of this accelerated aging syndrome. This work was supported by Fundación “la Caixa” (grant number LCF/PR/HR17/52150007 to VF, and JJF). JJF is supported by a Ramón y Cajal award (RYC2016–20026) from the Spanish Ministerio de Ciencia e Innovación (MICIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033 and Fondo Social Europeo “El FSE invierte en tu futuro”. VA’s lab is supported by MICIN/ AEI/10.13039/501100011033 and Fondo Social Europeo “El FSE invierte en tu futuro” (grant number PID2019-108489RBI00), the Progeria Research Foundation (Award PRF 2019–77), and a donation from Asociación Progeria Alexandra Peraut. LBG is supported by The Progeria Research Foundation. MDD is supported by a predoctoral FPI fellowship from the Spanish MICIN/AEI/10.13039/501100011033 and Fondo Social Europeo “El FSE invierte en tu futuro” (PRE2019-087463), and MA-P is supported by a predoctoral FPU contract from the Ministerio de Educación, Cultura y Deporte (FPU18/02913). The CNIC is supported by the MICIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant number CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

Humanα2-macroglobulin (hα2M) is a multidomain protein with a plethoraof essential functions, including transport of signaling molecules and endopeptidaseinhibition in innate immunity. Here, we dissected the molecular mechanism of theinhibitory function of the∼720-kDa hα2M tetramer through eight cryo–electronmicroscopy (cryo-EM) structures of complexes from human plasma. In the native com-plex, the hα2M subunits are organized in twoflexible modules in expanded conforma-tion, which enclose a highly porous cavity in which the proteolytic activity ofcirculating plasma proteins is tested. Cleavage of bait regions exposed inside the cavitytriggers rearrangement to a compact conformation, which closes openings and entrapsthe prey proteinase. After the expanded-to-compact transition, which occurs indepen-dently in the four subunits, the reactive thioester bond triggers covalent linking of theproteinase, and the receptor-binding domain is exposed on the tetramer surface forreceptor-mediated clearance from circulation. These results depict the molecular mecha-nism of a unique suicidal inhibitory trap., Thiswork was supported by grants from the Spanish Ministries of Economy and Com-petitivity (Grant No. BFU2017-88736-R) and of Science and Innovation (GrantNo. PID2020-113287RB-I00) and the Comunidad Aut onoma de Madrid (GrantNo. P2018/NMT-4389) to J.R.C. and by grants from Catalan and Spanish publicand private agencies (Grant Nos. BFU2019-107725-RB-I00 and 2017SGR00003;Fundaci o“La Marat odeTV3”Grant No. 201815) to F.X.G.-R. T.G. acknowledgesa Juan de la Cierva research contract (JCI-2012-13573) from the Ministry ofEconomy (MINECO) and S.R.M. a Ph.D.-fellowship (BES2016-076877) from theMinistry of Science and Innovation. The Structural Biology Unit of the MolecularBiology Institute of Barcelona (IBMB) was a Mar ıa de Maeztu Unit of Excellence(2015 to 2019) and the Centro Nacional de Biotecnolog ıa is a Severo OchoaCenter of Excellence (MINECO award SEV 2017-0712), as awarded by the Span-ish Ministry of Economy, Industry and Competitiveness.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells. NM has been funded by SNF grant 320030E-164245 and ERC Consolidator grant 2018 819717. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Benoît Zuber is supported by SNF grant 179520 and ERA-NET NEURON grant 185536. M.O. was supported by SNF grant PCEFP3_186993. Sí