ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs

Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-β-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.European Joint Program for Rare Diseases (EJPRD2019-40)Spanish Ministry of Science and Innovation and Universities (PGC2018-096049-B-I00)Junta de Andalucía (BIO-198, US-1254317 and US-1257019

Structural insights into the intracellular region of the human magnesium transport mediator CNNM4

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.Gobierno Vasco PI2010-17, IE05-147, IE07-202Diputación Foral de Vizcaya 7/13/08/2006/11, 7/13/08/2005/14Ministerio de Ciencia e Innovación BFU2010-17857, CSD2008-00005Ministerio de Economía y Competitividad BFU2013-47531-R, BES-2014-068464, BFU2016-77408-R, BES-2017-08043

Vascular and cognitive effects of cocoa-rich chocolate in postmenopausal women: a study protocol for a randomised clinical trial

Introduction The intake of polyphenols has certain health benefits. This study will aim to assess the effect of adding a daily amount of chocolate high in cocoa content and polyphenols to the normal diet on blood pressure, vascular function, cognitive performance, quality of life and body composition in postmenopausal women. Methods and analysis Here we plan a randomised clinical trial with two parallel groups involving a total of 140 women between 50 and 64 years in the postmenopausal period, defined by amenorrhoea of at least 12 consecutive months. The main variable will be the change in blood pressure. Secondary variables will be changes in vascular function, quality of life, cognitive performance and body composition. The intervention group will be given chocolate containing 99% cocoa, with instructions to add 10 g daily to their normal diet for 6 months. The daily nutritional contribution of this amount of chocolate is 59 kcal and 65.4 mg of polyphenols. There will be no intervention in the control group. All variables will be measured at the baseline visit and 3 and 6 months after randomisation, except cognitive performance and quality of life, which will only be assessed at baseline and at 6 months. Recruitment is scheduled to begin on 1 June 2018, and the study will continue until 31 May 2019. Ethics and dissemination This study was approved by the Clinical Research Ethics Committee of the Health Area of Salamanca, Spain (‘CREC of Health Area of Salamanca’), in February 2018. A SPIRIT checklist is available for this protocol. The clinical trial has been registered at ClinicalTrials. gov provided by the US National Library of Medicine, number NCT03492983. The results will be disseminated through open access peer-reviewed journals, conference presentations, broadcast media and a presentation to stakeholders.Gerencia Regional de Castilla y León (GRS 1583/B/1

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.Acknowledgements: This work was supported by Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (to MLM-C), Ministerio de Ciencia e Innovación CONSOLIDER-INGENIO 2010 Program Grant CSD2008-00005 (to LAMC); Spanish Ministry of Economy and Competitiveness Grant BFU2013-47531-R, BFU2016-77408-R, PID2019-109055RB-100 (to L.A.M.-C.) (MINECO/FEDER, UE); Asociación Española contra el Cáncer (MLM-C, TC-D), Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Fundacion BBVA UMBRELLA project (to M.L.M.-C.), Ayuda RYC2020-029316-I financiada por MICIN/AEI/10.13039/501100011033 (to TC-D), Plataforma de Investigación Clínica-SCReN (PT17 0017 0020) (to M.I.-L.), programa retos RTC2019-007125-1 (to M.L.M.-C, J.S.), Proyectos Investigacion en Salud DTS20/00138 (to M.L.M.-C., J.S), ERA-Net E-Rare EJP RD Joint Translational Call for Rare Diseases FIGHT-CNNM2 (EJPRD19-040) and from Instituto Carlos III, Spain (REF G95229142) (to L.A.M.-C.), US National Institutes of Health under grant CA217817 (to D.B.), Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thankMINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644) and PhD fellowship fromMINECO (REF BES-2017-080435) awarded to I.G.-R. The collection and storage of patients tissues was supported by the Newcastle Biomedicine Biobank and the European Community’s Seventh Framework Programme (FP7/2001–2013) and Cancer Research UK awards Cancer Research UK grants C18342/A23390; C9380/A18084 and C9380/A26813. Finally, we would like to acknowledge Begoña Rodríguez Iruretagoyena for the technical support provided

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals

Background and aims: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. Approach and results: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. Conclusions: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.Funding information: Supported by grants from Ministerio de Ciencia, Innovación y Universidades MICINN (PID2020-117116RB-100, RTI2018-096759-A-100, RTI2018-095114-B-I00, PID2019-108977RB-100 and RTI2018-095700-B100, integrado en el Plan Estatal de Investigación Científica y Técnica y Innovación, cofinanciado con Fondos FEDER, to M.L.M.-C., T.C.D., C.P., P.M.-S., and N.G.A.A., respectively), Subprograma Retos Colaboración RTC2019-007125-1; Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.); Asociación Española contra el Cáncer (to T.C.D. and M.S.-M); La Caixa Foundation Program (HR17-00601, to M.L.M.-C.), Proyectos Investigación en Salud DTS20/00138 (to M.L.M.-C.); Departamento de Industria del Gobierno Vasco (to M.L.M.-C.); Departamento de Educación del Gobierno Vasco (to N.G.-U. and J.S.); Acción Estratégica Ciber Emergentes 2018 (Ciberehd-ISCIII) and Gilead Sciences International Research Scholars Program in Liver Disease (to M.V.-R.); Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos IIIAcknowledgments: We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). We acknowledge Begoña Rodríguez Iruretagoyena for the technical support provided

Analysis of the impact of social determinants and primary care morbidity on population health outcomes by combining big data: A research protocol

In recent years, different tools have been developed to facilitate analysis of social determinants of health (SDH) and apply this to health policy. The possibility of generating predictive models of health outcomes which combine a wide range of socioeconomic indicators with health problems is an approach that is receiving increasing attention. Our objectives are twofold: (1) to predict population health outcomes measured as hospital morbidity, taking primary care (PC) morbidity adjusted for SDH as predictors; and (2) to analyze the geographic variability of the impact of SDH-adjusted PC morbidity on hospital morbidity, by combining data sourced from electronic health records and selected operations of the National Statistics Institute (Instituto Nacional de Estadística/INE).MethodsThe following will be conducted: a qualitative study to select socio-health indicators using RAND methodology in accordance with SDH frameworks, based on indicators published by the INE in selected operations; and a quantitative study combining two large databases drawn from different Spain’s Autonomous Regions (ARs) to enable hospital morbidity to be ascertained, i.e., PC electronic health records and the minimum basic data set (MBDS) for hospital discharges. These will be linked to socioeconomic indicators, previously selected by geographic unit. The outcome variable will be hospital morbidity, and the independent variables will be age, sex, PC morbidity, geographic unit, and socioeconomic indicators.AnalysisTo achieve the first objective, predictive models will be used, with a test-and-training technique, fitting multiple logistic regression models. In the analysis of geographic variability, penalized mixed models will be used, with geographic units considered as random effects and independent predictors as fixed effects.DiscussionThis study seeks to show the relationship between SDH and population health, and the geographic differences determined by such determinants. The main limitations are posed by the collection of data for healthcare as opposed to research purposes, and the time lag between collection and publication of data, sampling errors and missing data in registries and surveys. The main strength lies in the project’s multidisciplinary nature (family medicine, pediatrics, public health, nursing, psychology, engineering, geography)

Multiple health behaviour change primary care intervention for smoking cessation, physical activity and healthy diet in adults 45 to 75 years old (EIRA study): a hybrid effectiveness-implementation cluster randomised trial

Methods: A cluster randomised effectiveness-implementation hybrid trial-type 2 with two parallel groups was conducted in 25 Spanish Primary Health Care (PHC) centres (3062 participants): 12 centres (1481 participants) were randomised to the intervention and 13 (1581 participants) to the control group (usual care). The intervention was based on the Transtheoretical Model and focused on all target behaviours using individual, group and community approaches. PHC professionals made it during routine care. The implementation strategy was based on the Consolidated Framework for Implementation Research (CFIR). Data were analysed using generalised linear mixed models, accounting for clustering. A mixed-methods data analysis was used to evaluate implementation outcomes (adoption, acceptability, appropriateness, feasibility and fidelity) and determinants of implementation success. Results: 14.5% of participants in the intervention group and 8.9% in the usual care group showed a positive change in two or all the target behaviours. Intervention was more effective in promoting dietary behaviour change (31.9% vs 21.4%). The overall adoption rate by professionals was 48.7%. Early and final appropriateness were perceived by professionals as moderate. Early acceptability was high, whereas final acceptability was only moderate. Initial and final acceptability as perceived by the participants was high, and appropriateness moderate. Consent and recruitment rates were 82.0% and 65.5%, respectively, intervention uptake was 89.5% and completion rate 74.7%. The global value of the percentage of approaches with fidelity ≥50% was 16.7%. Eight CFIR constructs distinguished between high and low implementation, five corresponding to the Inner Setting domain. Conclusions: Compared to usual care, the EIRA intervention was more effective in promoting MHBC and dietary behaviour change. Implementation outcomes were satisfactory except for the fidelity to the planned intervention, which was low. The organisational and structural contexts of the centres proved to be significant determinants of implementation effectiveness

Methionine Cycle Rewiring by Targeting miR-873-5p Modulates Ammonia Metabolism to Protect the Liver from Acetaminophen

Drug-induced liver injury (DILI) development is commonly associated with acetaminophen (APAP) overdose, where glutathione scavenging leads to mitochondrial dysfunction and hepatocyte death. DILI is a severe disorder without effective late-stage treatment, since N-acetyl cysteine must be administered 8 h after overdose to be efficient. Ammonia homeostasis is altered during liver diseases and, during DILI, it is accompanied by decreased glycine N-methyltransferase (GNMT) expression and S-adenosylmethionine (AdoMet) levels that suggest a reduced methionine cycle. Anti-miR-873-5p treatment prevents cell death in primary hepatocytes and the appearance of necrotic areas in liver from APAP-administered mice. In our study, we demonstrate a GNMT and methionine cycle activity restoration by the anti-miR-873-5p that reduces mitochondrial dysfunction and oxidative stress. The lack of hyperammoniemia caused by the therapy results in a decreased urea cycle, enhancing the synthesis of polyamines from ornithine and AdoMet and thus impacting the observed recovery of mitochondria and hepatocyte proliferation for regeneration. In summary, anti-miR-873-5p appears to be an effective therapy against APAP-induced liver injury, where the restoration of GNMT and the methionine cycle may prevent mitochondrial dysfunction while activating hepatocyte proliferative response.We thank Ministerio de Ciencia e Innovación, Programa Retos-Colaboración RTC2019- 007125-1 (for J.S. and M.L.M.-C.); Instituto de Salud Carlos III: Proyectos de Investigación en Salud DTS20/00138 (for J.S. and M.L.M.-C.), PI20/00690 (for R.J.) and PT20/000127 (for M.I.L.); CIBERehd: EHD21TRF01/2022 (to M.L.M.-C.); Departamento de Industria del Gobierno Vasco (for M.L.M.-C.); Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 and RTI2018- 096759-1-100 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (for M.L.M.-C. and T.C.D., respectively); BIOEF (Basque Foundation for Innovation and Health Research); Asociación Española contra el Cáncer (AECC) (to M.L.M.-C., T.C.D.); AECC: GCTRA18006CARR (to A.C.); Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (for M.L.M.); La Caixa Foundation Program (for M.L.M.); BFU2015-70067-REDC, BFU2016-77408-R and BES-2017-080435 (MINECO/FEDER, UE); Ministerio de Ciencia, Innovación y universidades PID2019-108787RB-100 (to A.C.), PID2019- 109055RB-I00 (L.A.M.-C.), PID2020-117941RB-100 (to F.J.C.); Spanish Ministry of Economy and Competitiveness Grants BFU2013-47531-R and BFU2016-77408-R (L.A.M.-C.) and the FIGHT-CNNM2 project from the EJP RD Joint Transnational Call (JTC2019) (Ref. AC19/00073) (for L.A.M.-C.); Comunidad de Madrid: EXOHEP-CM S2017/BMD-3727 and NanoLiver-CM Y2018/NMT-4949 co-funded by European Structural and Investment Fund and COST Action CA17112 (to F.J.C.); Vencer el Cáncer Foundation (to A.C.); European Research Council: Consolidator Grant 819242 (to A.C.); CIBERONC and CIBERehd were funded by the Instituto de Salud Carlos III and Cofunded by FEDER funds. Partial funding for open access charge: Universidad de Málag

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury

Analysis of the impact of social determinants and primary care morbidity on population health outcomes by combining big data: A research protocol

BackgroundIn recent years, different tools have been developed to facilitate analysis of social determinants of health (SDH) and apply this to health policy. The possibility of generating predictive models of health outcomes which combine a wide range of socioeconomic indicators with health problems is an approach that is receiving increasing attention. Our objectives are twofold: (1) to predict population health outcomes measured as hospital morbidity, taking primary care (PC) morbidity adjusted for SDH as predictors; and (2) to analyze the geographic variability of the impact of SDH-adjusted PC morbidity on hospital morbidity, by combining data sourced from electronic health records and selected operations of the National Statistics Institute (Instituto Nacional de Estadística/INE).MethodsThe following will be conducted: a qualitative study to select socio-health indicators using RAND methodology in accordance with SDH frameworks, based on indicators published by the INE in selected operations; and a quantitative study combining two large databases drawn from different Spain’s Autonomous Regions (ARs) to enable hospital morbidity to be ascertained, i.e., PC electronic health records and the minimum basic data set (MBDS) for hospital discharges. These will be linked to socioeconomic indicators, previously selected by geographic unit. The outcome variable will be hospital morbidity, and the independent variables will be age, sex, PC morbidity, geographic unit, and socioeconomic indicators.AnalysisTo achieve the first objective, predictive models will be used, with a test-and-training technique, fitting multiple logistic regression models. In the analysis of geographic variability, penalized mixed models will be used, with geographic units considered as random effects and independent predictors as fixed effects.DiscussionThis study seeks to show the relationship between SDH and population health, and the geographic differences determined by such determinants. The main limitations are posed by the collection of data for healthcare as opposed to research purposes, and the time lag between collection and publication of data, sampling errors and missing data in registries and surveys. The main strength lies in the project’s multidisciplinary nature (family medicine, pediatrics, public health, nursing, psychology, engineering, geography)