The importance of the urea cycle and its relationships to polyamine metabolism during ammonium stress in Medicago truncatula

The ornithine–urea cycle (urea cycle) makes a signifcant contribution to the metabolic responses of lower photosynthetic eukaryotes to episodes of high nitrogen availability. In this study, we compared the role of the plant urea cycle and its relationships to polyamine metabolism in ammonium-fed and nitrate-fed Medicago truncatula plants. High ammonium resulted in the accumulation of ammonium and pathway intermediates, particularly glutamine, arginine, ornithine, and putrescine. Arginine decarboxylase activity was decreased in roots, suggesting that the ornithine decarboxylase-dependent production of putrescine was important in situations of ammonium stress. The activity of copper amine oxidase, which releases ammonium from putrescine, was signifcantly decreased in both shoots and roots. In addition, physiological concentrations of ammonium inhibited copper amine oxidase activity in in vitro assays, supporting the conclusion that high ammonium accumulation favors putrescine synthesis. Moreover, early supplementation of plants with putrescine avoided ammonium toxicity. The levels of transcripts encoding urea-cyclerelated proteins were increased and transcripts involved in polyamine catabolism were decreased under high ammonium concentrations. We conclude that the urea cycle and associated polyamine metabolism function as important protective mechanisms limiting ammonium toxicity in M. truncatula. These fndings demonstrate the relevance of the urea cycle to polyamine metabolism in higher plants.This work was supported by the grants from the Spanish Government AGL2014-52396-P (MICINN) and AGL2017-86293-P (MINECO/ FEDER) to JFM, and the Basque Government, Spain, IT-1018-16 (UPV/EHU-GV) to RE. MU is a recipient of a pre-doctoral fellowship from the Government of Navarre, Spain. JB and PLG have received pre-doctoral fellowships from the Public University of Navarre, Spain. PT has received funding from the Italian Ministry of Education, University and Research (Grant to Department of Science, University ‘Roma Tre’-‘Dipartimenti di Eccellenza’, ARTICOLO 1, COMMI 314– 337. LEGGE 423 232/2016; PRIN 2017—CUP F84I19000730005). Partial support was obtained from the Spanish State Research Agency AGL2017-83358-R (AEI/FEDER) and the Government of Aragon, Spain, Group A09-20R to YG. Open Access funding was provided by the Public University of Navarra

Enhanced mitochondrial activity reshapes a gut microbiota profile that delays NASH progression

[EN] Background and Aims: Recent studies suggest that mitochondrial dysfunction promotes progression to NASH by aggravating the gut-liver status. However, the underlying mechanism remains unclear. Herein, we hypothesized that enhanced mitochondrial activity might reshape a specific microbiota signature that, when transferred to germ-free (GF) mice, could delay NASH progression. Approach and Results: Wild-type and methylation-controlled J protein knockout (MCJ-KO) mice were fed for 6 weeks with either control or a choline-deficient, L-amino acid–defined, high-fat diet (CDA-HFD). One mouse of each group acted as a donor of cecal microbiota to GF mice, who also underwent the CDA-HFD model for 3 weeks. Hepatic injury, intestinal barrier, gut microbiome, and the associated fecal metabolome were then studied. Following 6 weeks of CDA-HFD, the absence of methylation-controlled J protein, an inhibitor of mitochondrial complex I activity, reduced hepatic injury and improved gut-liver axis in an aggressive NASH dietary model. This effect was transferred to GF mice through cecal microbiota transplantation. We suggest that the specific microbiota profile of MCJ-KO, characterized by an increase in the fecal relative abundance of Dorea and Oscillospira genera and a reduction in AF12, Allboaculum, and [Ruminococcus], exerted protective actions through enhancing short-chain fatty acids, nicotinamide adenine dinucleotide (NAD+) metabolism, and sirtuin activity, subsequently increasing fatty acid oxidation in GF mice. Importantly, we identified Dorea genus as one of the main modulators of this microbiota-dependent protective phenotype. Conclusions: Overall, we provide evidence for the relevance of mitochondria–microbiota interplay during NASH and that targeting it could be a valuable therapeutic approach.S

Early-Stage Breast Cancer Detection in Breast Milk

Breast cancer; Breast milkCáncer de mama; Leche maternaCàncer de mama; Llet maternaBreast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. Significance: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image.We thank the patients who participated in the study and donated samples for analysis for their generous contribution, with particular thanks to the first patient, Maite, and her daughter Àneu, who inspired us to initiate this study (oral consent to name the patient and her daughter was provided by the patient, and her legal partner provided written consent after patient's exitus). We are grateful to Javier Carmona for his valuable contributions and support in the manuscript's conceptualization, preparation, and revision. VHIO would like to acknowledge the Cellex Foundation for providing research facilities and equipment and the CERCA Programme from the Generalitat de Catalunya for their support of this research. The authors from VHIO acknowledge the State Agency for Research (Agencia Estatal de Investigación) for the financial support as a Center of Excellence Severo Ochoa (CEX2020-001024-S/AEI/10.13039/501100011033). This research is financially supported by the “El paseíco de la mama” Foundation. C. Saura was the recipient of a II FERO-GHD grant from the FERO Foundation (FERO/5086), a Junior Clinical award from the Spanish Association Against Cancer Foundation (FAECC; CLJUN212026ORTI), and a SEOM-Daiichi Sankyo grant for its support on the Breast Cancer Research Projects 2021 (SEOM/FECMA2022) and received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00198) and from the Instituto de Salud Carlos III (ISCIII) and Fondo Europeo de Desarrollo Regional (FEDER), cofunded by the European Union (PI21/01020). C. Ortiz was the recipient of a Junior Clinician award from the FAECC (CLJUN212026ORTI) and a SEOM-Daiichi Sankyo grant for its support on the Breast Cancer Research Projects 2021 (SEOM/FECMA2022), and received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00198). N. Bayó-Puxan received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00205), MCIN/AEI/10.13039/501100011033 (GPE2022-001029) and MCIN/AEI/10.130.39/501100011033, and the European Union “Next GenerationEU/PRTR” (ECT2020-000827). J.M. Miquel received funding from the Department of Health (Generalitat de Catalunya SLT008/18/00205), MCIN/AEI/10.130.39/501100011033, and the European Union “Next GenerationEU/PRTR” (ECT2020-000827). J. Arribas is funded by the Breast Cancer Research Foundation (BCRF-23-008), Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449, and PI22/00001), and the European Commission under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER and Asociación Española Contra el Cáncer. A. Vivancos was the recipient of a project award from the FAECC (AVP/18/AECC/3219) and received funding from the Advanced Molecular Diagnostic (DIAMAV) program from the FERO Foundation (8361) and from ISDIN for supporting the development of liquid biopsy applications at the Cancer Genomics Lab (1848). M. Sansó was the recipient of a II FERO-GHD grant from the FERO Foundation (FERO/5086) and an investigator award from the FAECC (INVES19056SANS), and received funding from the Health Research Institute of the Balearic Islands (IdISBa), the RADIX-Janssen program (RADIX/JANSSEN21/01), and the Miguel Servet Program funded by the ISCIII (CP22/00131)

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

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness

Un ataque combinado químico, virológico, biofísico y estructural hace posible la obtención de nuevos inhibidores de entrada celular de SARS-CoV-2 y la caracterización de su mecanismo de inhibición

Resumen del trabajo presentado al 45º Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Zaragoza del 5 al 8 de septiembre de 2023.IBV-COVID19 Pipeline: C.Espinosa, N.Gougeard, M.P.Hernández-Sierra, A.Rubio-del-Campo, R.Ruiz-Partida, L.Villamayor.El virus SARS-CoV-2 causa el COVID-19 al infectar las células a través de la interacción de la proteína de su espícula (S) con el receptor celular enzima convertidora de angiotensina 2 (ACE2). Para buscar inhibidores de este paso clave en la infección viral, examinamos una biblioteca interna (IQM-CSIC, Madrid) de compuestos multivalentes derivados de triptófano, primero usando pseudopartículas de Virus de Estomatits Vesicular que expresaban S (I2SysBio, UV y CSIC, Valencia), identificando un compuesto como potente inhibidor de entrada no citotóxico. La optimización química (IQM-CSIC) generó otros dos potentes inhibidores de entrada no citotóxicos que, como 2, también inhibieron la entrada celular de SARS-CoV-2 genuino (I2SysBio). Los estudios con proteínas recombinantes puras (IBV-CSIC, Valencia) usando termofluor y termoforesis de microescala revelaron la unión de estos compuestos a S, y a su dominio de unión al receptor producido separadamente, probando interferencia con la interacción con ACE2. La criomicroscopía electrónica de S (IBV-CSIC), libre o unido al compuesto activo, arrojó luz sobre los mecanismos de inhibición por estos compuestos de la entrada viral a la célula. Esta actividad triinstitucional combinada ha identificado y caracterizado una nueva clase de inhibidores de entrada de SARS-CoV-2 de claro potencial preventivo o terapéutico de COVID-19.ECNextGeneration EUfund 2020/2094 de CSIC/PTI Salud Global; Crue/CSIC/Santander Fondo Supera Covid-19;CSIC-COV19-082; CIBERER-ISCIIICOV20/00437; Covid19-SCI/GValenciana (RG);PID2020- 120322RB-C21 (VR) y PID2020-116880GB-I00 (JLLl) Agenc. Estat Investig.Peer reviewe

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.This work was supported by grants from Ministerio de Ciencia e Innovación, Programa Retos-Colaboración RTC2019-007125-1 (for Jorge Simon and Maria Luz Martinez-Chantar); Ministerio de Economía, Industria y Competitividad, Retos a la Sociedad AGL2017- 86927R (for F.M.); Instituto de Salud Carlos III, Proyectos de Investigación en Salud DTS20/00138 and DTS21/00094 (for Jorge Simon and Maria Luz Martinez-Chantar, and Asis Palazon. respectively); Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias co-founded by European Regional Development Fund/European Social Fund, “Investing in your future” PI19/00819, “Una manera de hacer Europa” FIS PI20/00765, and PI21/01067 (for Jose J. G. Marin., Pau Sancho-Bru,. and Mario F. Fraga respectively); Departamento de Industria del Gobierno Vasco (for Maria Luz Martinez-Chantar); Asturias Government (PCTI) co-funding 2018-2023/ FEDER IDI/2021/000077 (for Mario F. Fraga.); Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00, CEX2021-001136-S PID2020-117941RB-I00, PID2020-11827RB-I00 and PID2019-107956RA-100 integrado en el Plan Estatal de Investigación Científica y Técnica y Innovación, cofinanciado con Fondos FEDER (for Maria Luz Martinez-Chantar, Francisco J Cubero., Yulia A Nevzorova and Asis Palazon); Ayudas Ramón y Cajal de la Agencia Estatal de Investigación RY2013-13666 and RYC2018- 024183-I (for Leticia Abecia and Asis Palazon); European Research Council Starting Grant 804236 NEXTGEN-IO (for Asis Palazon); The German Research Foundation SFB/TRR57/P04, SFB1382-403224013/ A02 and DFG NE 2128/2-1 (for Francisco J Cubero and Yulia A Nevzorova); National Institute of Health (NIH)/National Institute of Alcohol Abuse and Alcoholism (NIAAA) 1U01AA026972-01 (For Pau Sancho-Bru); Junta de Castilla y León SA074P20 (for Jose J. G. Marin); Junta de Andalucía, Grupo PAIDI BIO311 (for Franz Martin); CIBERER Acciones Cooperativas y Complementarias Intramurales ACCI20-35 (for Mario F. Fraga); Ministerio de Educación, Cultura y Deporte FPU17/04992 (for Silvia Ariño); Fundació Marato TV3 201916-31 (for Jose J. G. Marin.); Ainize Pena-Cearra is a fellow of the University of the Basque Country (UPV/ EHU); BIOEF (Basque Foundation for Innovation and Health Research); Asociación Española contra el Cáncer (Maria Luz Martinez-Chantar and Teresa C. Delgado.); Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (for Maria Luz Martinez-Chantar); La Caixa Foundation Program (for Maria Luz Martinez-Chantar); Proyecto Desarrollo Tecnologico CIBERehd (for Maria Luz Martinez-Chantar); Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III.S

Effectiveness and safety of anti-CGRP monoclonal antibodies in patients over 65 years: a real-life multicentre analysis of 162 patients

Background Anti-CGRP monoclonal antibodies have shown notable effectiveness and tolerability in migraine patients; however, data on their use in elderly patients is still lacking, as clinical trials have implicit age restrictions and real-world evidence is scarce. In this study, we aimed to describe the safety and effectiveness of erenumab, galcanezumab and fremanezumab in migraine patients over 65 years old in real-life. Methods In this observational real-life study, a retrospective analysis of prospectively collected data from 18 different headache units in Spain was performed. Migraine patients who started treatment with any anti-CGRP monoclonal antibody after the age of 65 years were included. Primary endpoints were reduction in monthly migraine days after 6 months of treatment and the presence of adverse effects. Secondary endpoints were reductions in headache and medication intake frequencies by months 3 and 6, response rates, changes in patient-reported outcomes and reasons for discontinuation. As a subanalysis, reduction in monthly migraine days and proportion of adverse effects were also compared among the three monoclonal antibodies. Results A total of 162 patients were included, median age 68 years (range 65-87), 74.1% women. 42% had dyslipidaemia, 40.3% hypertension, 8% diabetes, and 6.2% previous cardiovascular ischaemic disease. The reduction in monthly migraine days at month 6 was 10.17.3 days. A total of 25.3% of patients presented adverse effects, all of them mild, with only two cases of blood pressure increase. Headache and medication intake frequencies were significantly reduced, and patient-reported outcomes were improved. The proportions of responders were 68%, 57%, 33% and 9% for reductions in monthly migraine days >= 30%,>= 50%,>= 75% and 100%, respectively. A total of 72.8% of patients continued with the treatment after 6 months. The reduction in migraine days was similar for the different anti-CGRP treatments, but fewer adverse effects were detected with fremanezumab (7.7%). Conclusions Anti-CGRP mAbs are safe and effective treatments in migraine patients over 65 years old in real-life clinical practice

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO