A Metabolomics Signature Linked To Liver Fibrosis In The Serum Of Transplanted Hepatitis C Patients

Liver fibrosis must be evaluated in patients with hepatitis C virus (HCV) after liver transplantation because its severity affects their prognosis and the recurrence of HCV. Since invasive biopsy is still the gold standard to identify patients at risk of graft loss from rapid fibrosis progression, it becomes crucial the development of new accurate, non-invasive methods that allow repetitive examination of the patients. Therefore, we have developed a non-invasive, accurate model to distinguish those patients with different liver fibrosis stages. Two hundred and three patients with HCV were histologically classified (METAVIR) into five categories of fibrosis one year after liver transplantation. In this cross-sectional study, patients at fibrosis stages F0-F1 (n = 134) were categorised as "slow fibrosers" and F2-F4 (n = 69) as "rapid fibrosers". Chloroform/methanol serum extracts were analysed by reverse ultra-high performance liquid chromatography coupled to mass spectrometry. A diagnostic model was built through linear discriminant analyses. An algorithm consisting of two sphingomyelins and two phosphatidylcholines accurately classifies rapid and slow fibrosers after transplantation. The proposed model yielded an AUROC of 0.92, 71% sensitivity, 85% specificity, and 84% accuracy. Moreover, specific bile acids and sphingomyelins increased notably along with liver fibrosis severity, differentiating between rapid and slow fibrosers

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.Acknowledgements: We thank Petros Tyrakis and Iván Martínez-Forero for critical reading and editing of the manuscript. Support was provided by the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the SPRI I+D COVID-19 fund (Gobierno Vasco). Personal fellowships: A.A.-V. (La Caixa Inphinit LCF/BQ/DR20/11790022), A.B. (AECC Bizkaia), A.G.d.R (Bikaintek), A.P. (Ramón y Cajal), B.J.-L. (Gob. Vasco), and E.P.-F. (Juan de la Cierva-Formación). M.L.M.-C. acknowledges RTC2019-007125-1, DTS20/00138, SAF2017-87301-R, and BBVA UMBRELLA project. M.L.-H. acknowledges the ISCIII for grant COV20-0170 and the Government of Cantabria for grant 2020UIC22-PUB-0019. O.M., J.-M.M., and N.G.A.A. acknowledge the Agencia Estatal de Investigación (Spain) for grants CTQ2015-68756-R, RTI2018-101269-BI00, and RTI2018-095700-B-I00, respectively. A.P. has received grant funding from the European Research Council (ERC), grant agreement number 804236 (Horizon 2020), and the FERO Foundation

Fatty liver and fibrosis in glycine N-methyltransferase knockout mice is prevented by nicotinamide

Deletion of glycine N-methyltransferase (GNMT) in mice, the main gene involved in liver S-adenosylmethionine (SAMe) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis. To demonstrate that the excess of hepatic SAMe is the main agent contributing to liver disease in GNMT-KO mice, we treated 1.5-month old GNMT-KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N-methyltransferase. NAM administration markedly reduced hepatic SAMe content, prevented DNA-hypermethylation and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis. More important, NAM treatment prevented the development of fatty liver and fibrosis in GNMT-KO mice. Because GNMT expression is down-regulated in patients with cirrhosis and there are subjects with GNMT mutations who have spontaneous liver disease, the clinical implication of the present findings is obvious at least with respect to these latter individuals. Especially since NAM has been used for many years to treat a broad spectrum of diseases including pellagra and diabetes without significant side effects, it should be considered in subjects with GNMT mutations.ConclusionsThese results indicate that the anomalous accumulation of SAMe in GNMT-KO mice can be corrected by NAM treatment leading to the normalization of the expression of many genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation and apoptosis, and to the reversion of the appearance of the pathologic phenotype

Sirtuin 1 regulation of developmental genes during differentiation of stem cells

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells

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

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

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation

12 Pág.In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.Projects PID2020-112723RB-I00 and PID2021-122507OB-I00 funded by MCIN1/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. José Manuel Ortiz Rodríguez was funded by PNR - DIMIVET- University of Bologna - CUP J45F21002000001, Ministry of University and Research (D.M. 737/2021). This work was supported by ‘Junta de Extremadura’ (Spain) and ‘Fondo Europeo de Desarrollo Regional’; Reference: IB20005. Marcos Luis Calero was supported by a grant “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación. Acción II” from the University of Extremadura (Ref. Beca RC1). Grant FJC2021-047675-I funded by MCIN2/AEI /10.13039/501100011033 and European Union NextGenerationEU/PRTR to Federica Marinaro. We appreciate the kind help of Paula Navarrete López for proteomic figures handling. The help of the veterinary staff of the INCARSA abbatoir is greatly acknowledged. This paper is dedicated to de memory of Dr. Juan Florencio Macías Núñez who seeded the curiosity and love for research in B. M-G.Peer reviewe

The metabolic co-regulator PGC1α suppresses prostate cancer metastasis

Cellular transformation and cancer progression is accompanied by changes in the metabolic landscape. Master co-regulators of metabolism orchestrate the modulation of multiple metabolic pathways through transcriptional programs, and hence constitute a probabilistically parsimonious mechanism for general metabolic rewiring. Here we show that the transcriptional co-activator peroxisome proliferator-activated receptor gamma co-activator 1α (PGC1α) suppresses prostate cancer progression and metastasis. A metabolic co-regulator data mining analysis unveiled that PGC1α is downregulated in prostate cancer and associated with disease progression. Using genetically engineered mouse models and xenografts, we demonstrated that PGC1α opposes prostate cancer progression and metastasis. Mechanistically, the use of integrative metabolomics and transcriptomics revealed that PGC1α activates an oestrogen-related receptor alpha (ERRα)-dependent transcriptional program to elicit a catabolic state and metastasis suppression. Importantly, a signature based on the PGC1α–ERRα pathway exhibited prognostic potential in prostate cancer, thus uncovering the relevance of monitoring and manipulating this pathway for prostate cancer stratification and treatment

Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

Background and Aims We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. Approach and Results We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6, and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. Conclusions Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.National Institutes of Health (R01DK123763, R01DK119437, HL151328, P30DK52574, P30DK56341, and UL1TR002345); Ministerio de Economía y Competitividad de España (SAF2017-88041-R); Ministerio de Economía y Competitividad de España for the Severo Ochoa Excellence Accreditation (SEV-2016-0644); CIBERehd (Biomedical Research Center in Hepatic and Digestive Diseases) and Netherlands Organization for Applied Scientific Research Program (PMC13 and PMC15); Spanish Carlos III Health Institute (PI15/01132 and PI18/01075); Miguel Servet Program (CON14/00129 and CPII19/00008); Fondo Europeo de Desarrollo Regional, CIBERehd, Department of Industry of the Basque Country (Elkartek: KK-2020/00008); La Caixa Scientific Foundation (HR17-00601); Liver Investigation: Testing Marker Utility in Steatohepatitis consortium funded by the Innovative Medicines Initiative Program of the European Union (777377), which receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA; Newcastle NIHR Biomedical Research Center; Czech Ministry of Health (RVO-VFN64165/2020); Fondo Nacional De Ciencia y Tecnología de Chile (1191145); and the Comisión Nacional de Investigación, Ciencia y Tecnología (AFB170005, CARE Chile UC); Agencia Nacional de Investigación y Desarrollo (ANID ACE 210009); European Union's Horizon 2020 Research and Innovation Program (825510)

Las proteínas RHO inducen propiedades metastasicas "in vivo"

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 10-10-1997Las proteínas Rho son GTPasas de bajo peso molecular que se encuentran en su estado activo cuando están unidas a GTP. Se ha involucrado a esta familia de proteínas en numerosos procesos dentro de la célula como regulación del citoesqueleto, formación de la yema en levaduras, motilidad celular, citoquinesis, contracción del músculo liso, agregación de plaquetas y linfocitos, transformación y apoptosis. Varios de los miembros de esta familia incluyendo el gen rho de Aplysia californicci y los genes humanos rho A, rho B y rac 1 son capaces de inducir transformación y células sobreexpresando estos genes son tumorigénicas cuando se inyectan en ratones desnudos. Existen evidencias de que estas proteínas podrían estar también involucradas en la inducción de metástasis aunque los datos aportados hasta ahora han sido realizados de manera indirecta o mediante experimentos in vitro. En este estudio se ha investigado si células transformadas por los oncogenes rlzo son capaces de inducir metástasis irz vivo. Para ello se inyectaron fibroblastos de ratón NIH 3T3 sobreexpresando el gen rho de Aplysia californica en la vena de la cola de ratones desnudos (ensayo de metástasis experimental), y se comprobó cómo eran capaces de colonizar mayoritariamente el pulmón así como otros órganos (tejido adiposo, ganglios linfáticos). En un segundo tipo de ensayo se inyectaron estas células subcutáneamente en la extremidad posterior de los ratones y posteriormente se eliminó el tumor primario formado (ensayo de metástasis espontánea). Se pudo comprobar como los tumores generados fueron capaces de metastatizar en distintos órganos, principalmente en pulmón y bazo. Estos datos se reprodujeron utilizando los genes humanos rho A y rac 1 en su versión activada así como los oncogenes dbl, vav y ost que son factores de intercambio para los miembros de la familia rho. Nuestros resultados demuestran que las vías de señalización reguladas por las proteínas Rho juegan un papel relevante en la adquisición del fenotipo metastásico irz vivo

Apoptosis Induced by Rac GTPase Correlates with Induction of FasL and Ceramides Production

Rho proteins, members of the Ras superfamily of GTPases, are critical elements in signal transduction pathways governing cell proliferation and cell death. Different members of the family of human Rho GTPases, including RhoA, RhoC, and Rac1, participate in the regulation of apoptosis in response to cytokines and serum deprivation in different cell systems. Here, we have characterized the mechanism of apoptosis induced by Rac1 in NIH 3T3 cells. It requires protein synthesis and caspase-3 activity, but it is independent of the release of cytochrome c from mitochondria. Moreover, an increase in mitochondria membrane potential and the production of reactive oxygen species was observed. Rac1-induced apoptosis was related to the simultaneous increase in ceramide production and synthesis of FasL. Generation of FasL may be mediated by transcriptional regulation involving both c-Jun amino terminal kinase as well as nuclear factor-κB-dependent signals. None of these signals, ceramides or FasL, was sufficient to induce apoptosis in the parental cell line, NIH 3T3 cells. However, any of them was sufficient to induce apoptosis in the Rac1-expressing cells. Finally, inhibition of FasL signaling drastically reduced apoptosis by Rac1. Thus, Rac1 seems to induce apoptosis by a complex mechanism involving the generation of ceramides and the de novo synthesis of FasL. These results suggest that apoptosis mediated by Rac1 results from a signaling mechanism that involves biochemical and transcriptional events under control of Rac1