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We have developed two advanced cirrhosis regression experimental models with persistent relevant fibrosis and portal hypertension and an associated deteriorated metabolism that mimic what happens in patients. LI, despite improving metabolism, did not enhance the regression process in our cirrhotic models. CR did not further reduce PP, hepatic fibrosis, or HSC activation. MEE exhibited a profibrogenic effect in the liver blunting cirrhosis regression. One of the potential explanations of this worsening could be ammonia accumulation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a multi-organ damage that includes hepatic dysfunction, which has been observed in over 50% of COVID- 19 patients. Liver injury in COVID-19 could be attributed to the cytopathic effects, exacerbated immune responses or treatment-associated drug toxicity. Herein we demonstrate that hepatocytes are susceptible to infection in different models: primary hepatocytes derived from humanized angiotensin-converting enzyme-2 mice (hACE2) and primary human hepatocytes. Pseudotyped viral particles expressing the full-length spike of SARS-CoV-2 and recombinant receptor binding domain (RBD) bind to ACE2 expressed by hepatocytes, promoting metabolic reprogramming towards glycolysis but also impaired mitochondrial activity. Human and hACE2 primary hepatocytes, where steatosis and inflammation were induced by methionine and choline deprivation, are more vulnerable to infection. Inhibition of the reninangiotensin system increases the susceptibility of primary hepatocytes to infection with pseudotyped viral particles. Metformin, a common therapeutic option for hyperglycemia in type 2 diabetes patients known to partially attenuate fatty liver, reduces the infection of human and hACE2 hepatocytes. In summary, we provide evidence that hepatocytes are amenable to infection with SARS-CoV-2 pseudovirus, and we propose that metformin could be a therapeutic option to attenuate infection by SARS-CoV-2 in patients with fatty liver.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis., p53 regulates signalling pathways involved in metabolic homeostasis. Here the authors show that O-GlcNAcylation of p53 in the liver plays a key role in the physiological regulation of glucose homeostasis, potentially via controlling the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase.

Background and aims Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. Approach and results Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. Conclusions Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

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., Egia-Mendikute et al. develop a multiplexed flow cytometric bead array to simultaneously detect antibodies reactive to three immunogenic SARS-CoV-2 proteins. More sensitive than ELISA, they detected N-reactive antibodies in COVID-19-negative individuals with this assay, showing it to have superior potential to detect low antibody titres compared to current gold standard serology methods.

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2- mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis.

17 páginas, 6 figuras, 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.

[Background & Aims] Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to non-alcoholic fatty liver disease (NAFLD) remains unknown., [Methods] By performing proteomic analysis on livers from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice., [Results] ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and non-alcoholic steatohepatitis) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoyltransferase 1a (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action., [Conclusions] Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis., This work has been supported by grants from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (PA: RTI2018-095134-B-100; DS and LH: SAF2017-83813-C3-1-R; MLMC: RTC2019-007125-1; CD: BFU2017-87721; ML: RTI2018–101840-B-I00; GS; PID2019-104399RB-I00; RN: RTI2018-099413-B-I00 and RED2018-102379-T; MLMC: SAF2017-87301-R; TCD: RTI2018-096759-A-100), FEDER/Instituto de Salud Carlos III (AGR: PI19/00123), Xunta de Galicia (ML: 2016-PG068; RN: 2015-CP080 and 2016-PG057), Fundación BBVA (RN, GS and MLM), Proyectos Investigación en Salud (MLMC: DTS20/00138), Sistema Universitario Vasco (PA: IT971-16); Fundación Atresmedia (ML and RN), Fundación La Caixa (M.L., R.N. and M.C.), Gilead Sciences International Research Scholars Program in Liver Disease (MVR), Marató TV3 Foundation (DS: 201627), Government of Catalonia (DS: 2017SGR278) and European Foundation for the Study of Diabetes (RN and GS). This research also received funding from the European Community’s H2020 Framework Programme (ERC Synergy Grant-2019-WATCH- 810331, to RN, VP and MS). Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem). CIBERobn, CIBERehd and CIBERdem are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644).

Neuroinvasion by SARS-CoV-2 is now accepted. To investigate whether low testosterone levels observed in men with severe COVID-19 could be of central origin, we retrospectively analyzed blood samples from 60 male intensive-care patients and explored SARS-CoV-2 brain entry using animal and cellular models as well as adult COVID-19 patient and fetal human brains. Most hypotestosteronemic patients displayed hypogonadotropic hypogonadism or abnormal hypothalamic-pituitary-gonadal axis regulation. Neurons producing gonadotropin-releasing hormone (GnRH), the master molecule controlling fertility, expressed angiotensin-converting enzyme 2 and neuropilin-1, two host-cell factors mediating infection, and were infected and dying in all COVID-19 patient brains. Tanycytes - hypothalamic glia that regulate GnRH secretion - were also infected. Additionally, human fetal olfactory and vomeronasal epithelia, from which GnRH neurons arise, richly expressed both the above host-cell susceptibility factors and formyl peptide receptor 2, a putative vomeronasal receptor that also appeared involved in SARS-CoV-2 pathogenesis in humans and mice. Finally, a fetal human GnRH cell line expressing all these receptors could be infected by a SARS-CoV-2-like pseudovirus. Together, our findings suggest that GnRH neurons, which may be implicated in brain development and aging in addition to reproduction, are particularly vulnerable to SARS-CoV-2 in both adults and fetuses/newborns, with potentially devastating long-term consequences.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a multi-organ damage that includes hepatic dysfunction, which has been observed in over 50% of COVID-19 patients. Liver injury in COVID-19 could be attributed to the cytopathic effects, exacerbated immune responses or treatment-associated drug toxicity. Herein we demonstrate that hepatocytes are susceptible to infection in different models: primary hepatocytes derived from humanized angiotensin-converting enzyme-2 mice (hACE2) and primary human hepatocytes. Pseudotyped viral particles expressing the full-length spike of SARS-CoV-2 and recombinant receptor binding domain (RBD) bind to ACE2 expressed by hepatocytes, promoting metabolic reprogramming towards glycolysis but also impaired mitochondrial activity. Human and hACE2 primary hepatocytes, where steatosis and inflammation were induced by methionine and choline deprivation, are more vulnerable to infection. Inhibition of the renin-angiotensin system increases the susceptibility of primary hepatocytes to infection with pseudotyped viral particles. Metformin, a common therapeutic option for hyperglycemia in type 2 diabetes patients known to partially attenuate fatty liver, reduces the infection of human and hACE2 hepatocytes. In summary, we provide evidence that hepatocytes are amenable to infection with SARS-CoV-2 pseudovirus, and we propose that metformin could be a therapeutic option to attenuate infection by SARS-CoV-2 in patients with fatty liver.

To the Editor: We read with great interest the article published by Biquard and colleagues showing that, according to public transcriptomic data, the hepatic expression of angiotensin converting enzyme 2 (ACE2) and the cellular transmembrane protease serine 2 (TMPRSS2) remains unchanged in patients with metabolicassociated fatty liver disease (MAFLD).1 SARS-CoV-2 attaches to cells by binding to its receptor ACE2. TMPRSS2 then cleaves the SARS-CoV-2 spike protein, allowing fusion of cellular and viral membranes.2 Despite this retrospective study, there is growing evidence that patients with MAFLD are at higher risk of COVID-19 disease progression.

Liver kinase B1 (LKB1) also referred to as serine/threonine kinase 11 (STK11) encodes a 50 kDa evolutionary conserved serine/threonine kinase that is ubiquitously expressed in adult and fetal tissues. LKB1 is a master kinase known to phosphorylate and activate several kinases including AMP-activated protein kinase, a crucial cellular energy sensor. LKB1 shows pleiotropic activity playing diverse roles in multiple processes, including cell polarity and other processes relevant in cancer pathology, such as energy metabolism, proliferation and apoptosis. In spite of the fact that LKB1 is often considered a tumor suppressor in a wide variety of organs, in the last years, several studies have shown that LKB1 is unexpectedly high in hepatocellular carcinoma (HCC), the most common type of primary liver cancer. Post-translational modifications of LKB1 are potentially relevant in HCC. Herein, we provide a comprehensive revision of post-translational modifications of LKB1 in HCC and how they modulate LKB1 function by different mechanisms such as regulation of its activity, localization or stability. Overall, the signature post-translational modifications of LKB1 in HCC appear to play an important role in the rather unique role of LKB1 as an oncogenic driver in liver cancer and may provide an alternative valuable therapeutic approach to regulate LKB1 expression and/or activity in HCC.

Although the multi-tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)-CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA-CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up-regulation in tumor tissue was found for miR-141 and miR-330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral-mediated transduction of eCCA (EGI-1 and TFK-1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up-regulated in CCA. Conclusion: The reason for impaired hOCT1-mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib.

Background & Aims: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). Methods: Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. Results: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. Lay summary: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein., Graphical Abstract

Dysregulation of miRNAs is a hallmark of cancer, modulating oncogenes, tumor suppressors, and drug responsiveness. The multi-kinase inhibitor sorafenib is one of the first-line drugs for advanced hepatocellular carcinoma (HCC), although the outcome for treated patients is heterogeneous. The identification of predictive biomarkers and targets of sorafenib efficacy are sorely needed. Thus, selected top upregulated miRNAs from the C19MC cluster were analyzed in different hepatoma cell lines compared to immortalized liver human cells, THLE-2 as control. MiR-518d-5p showed the most consistent upregulation among them. Thus, miR-518d-5p was measured in liver tumor/non-tumor samples of two distinct cohorts of HCC patients (n = 16 and n = 20, respectively). Circulating miR-518d-5p was measured in an independent cohort of HCC patients receiving sorafenib treatment (n = 100), where miR-518d-5p was analyzed in relation to treatment duration and patient’s overall survival. In vitro and in vivo studies were performed in human hepatoma BCLC3 and Huh7 cells to analyze the effect of miR-518d-5p inhibition/overexpression during the response to sorafenib. Compared with healthy individuals, miR-518d-5p levels were higher in hepatic and serum samples from HCC patients (n = 16) and in an additional cohort of tumor/non-tumor paired samples (n = 20). MiR-518d-5p, through the inhibition of c-Jun and its mitochondrial target PUMA, desensitized human hepatoma cells and mouse xenograft to sorafenib-induced apoptosis. Finally, serum miR-518d-5p was assessed in 100 patients with HCC of different etiologies and BCLC-stage treated with sorafenib. In BCLC-C patients, higher serum miR-518d-5p at diagnosis was associated with shorter sorafenib treatment duration and survival. Hence, hepatic miR-518d-5p modulates sorafenib resistance in HCC through inhibition of c-Jun/PUMA-induced apoptosis. Circulating miR-518d-5p emerges as a potential lack of response biomarker to sorafenib in BCLC-C HCC patients.

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo., Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (SAF2015-65327-R and RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB, AGL2017-86757-R to LA, SAF2017-87301-R to MLMC, SAF2015-64111-R to AP, SAF2015-73549-JIN to HR), Instituto de Salud Carlos III (PIE13/0004 to AP), the Basque Government Department of Health (2015111117 to LA), the Basque Foundation for Innovation and Health Research (BIOEF), through the EiTB Maratoia grant BIO15/CA/016/BS to MLMC, the regional Government of Andalusia co-funded by CEC and FEDER funds (Proyectos de Excelencia P12-CTS-2232) and Fundación Domingo Martínez (to AP). LA is supported by the Ramon y Cajal program (RYC-2013-13666). DB, MMR and TMM are recipients of MCIU FPI fellowships. ACG and AP are recipients of fellowships form the Basque Government. APC is a recipient of a fellowship from the University of the Basque Country. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia Province and the CIBERehd network. DB and JA are supported by a grant from the Jesús de Gangoiti Barrera Foundation.

Cystathionine β-synthase (CBS), the pivotal enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal-5’-phosphate-dependent condensation of serine with homocysteine to form cystathionine. Additionally, CBS performs alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide (H2S), an important signal transducer in many physiological and pathological processes. Toxoplasma gondii, the causative agent of toxoplasmosis, encodes a functional CBS (TgCBS) that contrary to human CBS, is not allosterically regulated by S-adenosylmethionine and can use both, Ser and O-acetylserine (OAS) as substrates. TgCBS is also strongly implicated in the production of H2S, and thus involved in redox homeostasis of the parasite. Here, we report its crystal structure, the first CBS from a protozoan described so far. Our data reveals a basal-like fold that unexpectedly differs from the active conformations found in other organisms, but structurally similar to the pathogenic activated mutant D444N of the human enzyme.

Non-alcoholic steatohepatitis (NASH) is characterized by an abnormal hepatic lipid accumulation accompanied by a necro-inflammatory process and a fibrotic response. It comprises from 10% to 30% of cases of patients with non-alcoholic liver disease, which is a global health problem affecting around a quarter of the worldwide population. Nevertheless, the development of NASH is often surrounded by a pathological context with other comorbidities, such as cardiovascular diseases, obesity, insulin resistance or type 2 diabetes mellitus. Dietary imbalances are increasingly recognized as the root cause of these NASH-related comorbidities. In this context, a growing concern exists about whether magnesium consumption in the general population is sufficient. Hypomagnesemia is a hallmark of the aforementioned NASH comorbidities, and deficiencies in magnesium are also widely related to the triggering of complications that aggravate NASH or derived pathologies. Moreover, the supplementation of this cation has proved to reduce mortality from hepatic complications. In the present review, the role of magnesium in NASH and related comorbidities has been characterized, unraveling the relevance of maintaining the homeostasis of this cation for the correct functioning of the organism.

Objective Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. Methods Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8. Results Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models Conclusions Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD., Graphical abstract Image 1, Highlights • Hepatic neddylation is induced in clinical and pre-clinical NAFLD. • Neddylation inhibition reduces hepatic steatosis in pre-clinical models of NAFLD. • Neddylation inhibition boosts fatty acid oxidation through a DEPTOR/mTOR axis. • Neddylation inhibition reduces hepatic inflammation and lipid peroxidation in NAFLD. • Serum NEDD8 levels correlate positively with liver disease in patients and pre-clinical models of NAFLD.

Background Polycystic liver diseases (PLDs) are genetic inherited disorders characterized by the progressive growth of numerous intrahepatic biliary cysts, which are the main cause of morbidity. Previous studies revealed that cystic cholangiocytes are characterized by endoplasmic reticulum stress and aberrant posttranslational modification (PTM) of proteins, in particular hyper-SUMOylation, that promote PLD pathobiology. Protein NEDDylation is a newly characterized PTM that modulates a plethora of biological processes and its dysregulation is associated with the development and progression of several human diseases. However, the role of NEDDylation in PLD remains elusive. Objective To explore the role of protein NEDDylation in PLD and its potential therapeutic regulatory value. Methods Levels and functional effects of NEDDylation, including response to Pevonedistat (first-in-class selective inhibitor of the NEDDylation E1 enzyme NAE), were assessed in vitro, in vivo, and/or in patients with PLD. NEDDylated protein levels in normal and cystic human cholangiocytes were assessed by immunoprecipitation, and the proteomic profile was further analyzed by mass spectrometry. Results and Conclusion The genes involved in the NEDDylation pathway were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture, compared to controls. Elevated levels of NEDDylated proteins were further confirmed in cystic cholangiocytes in vitro, which diminished under Pevonedistat incubation. Pevonedistat promoted apoptotic cell death and reduced proliferation in cystic cholangiocytes in vitro. Comparative proteomic profiling of NEDD8-immunoprecipitated proteins between normal and cystic cholangiocytes in culture reported candidate proteins involved in cystogenesis, mostly associated with protein biogenesis and quality control. All these data indicate that cystic cholangiocytes display increased protein NEDDylation, contributing to cell survival and proliferation, ultimately supporting hepatic cystogenesis. Targeting of protein hyper-NEDDylation in cystic cholangiocytes inhibits cystogenesis in experimental models, representing a novel therapeutic opportunity in PLD. Spanish Carlos III Health Institute (ISCIII), Grant/Award Numbers: CON14/00129, CPII19/00008, FIS PI12/00380, FIS PI14/ 00399, FIS PI15/01132, FIS PI17/00022, FIS PI18/01075, FIS PI20/00186, Sara Borrell CD19/00254; Diputacion Foral de Gipuzkoa, Grant/Award Numbers: DFG15/010, DFG16/004; Department of Health of the Basque Country, Grant/Award Numbers: 2015111100, 2017111010, 2019111024; Euskadi RIS3, Grant/Award Numbers: 2016222001, 2017222014, 2018222029, 2019222054, 2020333010; Department of Industry of the Basque Country, Grant/Award Number: KK-2020/00008; Spanish Ministry of Economy and Competitiveness, Grant/Award Number: RYC-2015-17755; Ministerio de Ciencia, Innovacion y Universidades, Grant/ Award Number: SAF2017-87301-R; Ayudas para apoyar grupos de investigacion del Sistema Universitario Vasco, Grant/Award Number: IT971-16; Universita Politecnica delle Marche, Grant/Award Number: PSA2017_UNIVPM; European Association for the Study of the Liver, Grant/Award Number: Sheila Sherlock Award 2017; Spanish Ministry of Science and Innovation, Grant/Award Number: BES-2014-069148; Basque Government, Grant/Award Number: PRE_2016_1_0269; Basque Foundation for Innovation and Health Research, Grant/Award Number: BIO15/CA/016/BD; Fundacion Cientifica de la Asociacion Espanola Contra el Cancer; La Caixa Scientific Foundation, Grant/ Award Number: HR17-00601; CIBERehd; Fondo Europeo de Desarrollo Regional Document

Background & Aims: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. Methods: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. Results: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdhl-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUM01-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. Conclusions: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy., Lay summary: Protein SUMOylation is a dynamic post-translational event implicated in numerous cellular processes. This study revealed dysregulated protein SUMOylation in polycystic liver disease, which promotes hepatic cystogenesis. Administration of S-adenosylmethionine (SAMe), a natural UBC9-dependent SUMOylation inhibitor, halted polycystic liver disease in experimental models, thus representing a potential therapeutic agent for patients., Graphical Abstract

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor &beta, (TGF&beta, ). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strategy.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process &ldquo, protein polyubiquitination&rdquo, is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome,specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis. Keywords: liver fibrosis; ubiquitination; metabolomics; proliferating cell nuclear antigen (PCNA); DNA damage response (DDR)

Current food tendencies, suboptimal dietary habits and a sedentary lifestyle are spreading metabolic disorders worldwide. Consequently, the prevalence of liver pathologies is increasing, as it is the main metabolic organ in the body. Chronic liver diseases, with non-alcoholic fatty liver disease (NAFLD) as the main cause, have an alarming prevalence of around 25% worldwide. Otherwise, the consumption of certain drugs leads to an acute liver failure (ALF), with drug-induced liver injury (DILI) as its main cause, or alcoholic liver disease (ALD). Although programs carried out by authorities are focused on improving dietary habits and lifestyle, the long-term compliance of the patient makes them difficult to follow. Thus, the supplementation with certain substances may represent a more easy-to-follow approach for patients. In this context, the consumption of polyphenol-rich food represents an attractive alternative as these compounds have been characterized to be effective in ameliorating liver pathologies. Despite of their structural diversity, certain similar characteristics allow to classify polyphenols in 5 groups: stilbenes, flavonoids, phenolic acids, lignans and curcuminoids. Herein, we have identified the most relevant compounds in each group and characterized their main sources. By this, authorities should encourage the consumption of polyphenol-rich products, as most of them are available in quotidian life, which might reduce the socioeconomical burden of liver diseases. We thank University of Basque Country (UPV/EHU), Basque Government and Asociacion Espanola Contra el Cancer (AECC) for the Pre-doctoral grants to M.C.-A., N.G.-U. and M.S.-M., respectively. Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644).

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process “protein polyubiquitination” is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.

BACKGROUND Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS Isolated primary mouse hepatocytes were incubated with 20 mu mol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with(13)C-uniformly labeled glucose. For thein vivopart of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACC alpha/beta(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with(13)C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P. CONCLUSION Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA beta-oxidation and oxidative phosphorylation. Supported by the National Institutes of Health Grant, No. R01CA172086; Plan Nacional of I+D, No. SAF2017-88041-R; Ministerio de Economia y Competitividad de Espana, No. SAF2017-87301-R; Asociacion Espanola contra el Cancer, No. AECC17/302; Ayudas Fundacion BBVA a equipos de Investigacion Cientifica 2018; Fondo Europeo de Desarrollo Regional, Ministerio de Economia y Competitividad de Espana, No. PGC2018-099857-BI00; Basque Government Grants, No. IT1264-19; Ministerio de Economia y Competitividad de Espana for the Severo Ochoa Excellence Accreditation, No. SEV2016-0644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

© 2020 by the authors., Prostate cancer is the most frequent malignancy in European men and the second worldwide. One of the major oncogenic events in this disease includes amplification of the transcription factor cMYC. Amplification of this oncogene in chromosome 8q24 occurs concomitantly with the copy number increase in a subset of neighboring genes and regulatory elements, but their contribution to disease pathogenesis is poorly understood. Here we show that TRIB1 is among the most robustly upregulated coding genes within the 8q24 amplicon in prostate cancer. Moreover, we demonstrate that TRIB1 amplification and overexpression are frequent in this tumor type. Importantly, we find that, parallel to its amplification, TRIB1 transcription is controlled by cMYC. Mouse modeling and functional analysis revealed that aberrant TRIB1 expression is causal to prostate cancer pathogenesis. In sum, we provide unprecedented evidence for the regulation and function of TRIB1 in prostate cancer., We are grateful to the Carracedo lab for valuable input, to Ana M. Aransay and F. Elortza for technical advice. P.S. was funded by the European Training Networks Project (H2020-MSCA-ITN-308 2016 721532). V.T. is funded by Fundación Vasca de Innovación e Investigación Sanitarias, BIOEF (BIO15/CA/052), the AECC J.P. Bizkaia, the Basque Department of Health (2016111109) and the MICINN RTI2018-097267-B-I00. A. Macchia was funded by a FPI predoctoral fellowship from MICINN (PRE2018-083607). EB is funded by the MICINN (BFU2016-76872-R (FEDER/EU), PID2019-108112RB-I00, Severo Ochoa Excellence Accreditation SEV-2016-0644 and Excellence Networks SAF2017-90794-REDT). The work of A. Carracedo is supported by the Basque Department of Industry, Tourism and Trade (Elkartek), the department of education (IKERTALDE IT1106-16) and health (RIS3), the BBVA foundation and the MICINN (SAF2016-79381-R (FEDER/EU; PID2019-108787RB-I00) Severo Ochoa Excellence Accreditation SEV-2016-0644 and Excellence Networks SAF2016-81975-REDT and RED2018-102769-T), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17/ and the European Research Council (Starting Grant 336343, Consolidator Grant 819242). CIBERONC was co-funded with FEDER funds and funded by ISCIII.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and the second most common cause of cancer mortality worldwide [...]

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD., Non-alcoholic fatty liver (NAFLD) disease causes degeneration of the liver, affects about 25% of people globally, and has no approved treatment. Here, the authors show that the therapeutic siRNA-driven silencing of MCJ in the liver is an effective and safe treatment for NAFLD in multiple mouse models.

Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n = 36) and malignant conditions, CCA (n = 36) or PDAC (n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease andidentifyingbiomarkersforthedifferentialdiagnosisofbiliarystrictures. Comprehensiveanalyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n = 10) and proteins (n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy. Keywords: human bile; cholangiocarcinoma; pancreatic adenocarcinoma; lipidomics; proteomics; machine-learning &nbsp

Background and purpose The expression of the human organic cation transporter-1 (hOCT1, gene SLC22A1) is reduced in hepatocellular carcinoma (HCC). The molecular bases of this reduction and its relationship with the poor response of HCC to sorafenib were investigated. Experimental approach HCC transcriptomes from 366 samples available at TCGA were analysed. Alternative splicing was determined by RT-PCR. The role of miRNAs in SLC22A1 downregulation was investigated. Expression of Oct1 was measured in rodent HCC models (spontaneously generated in Fxr-/- mice and chemically-induced in rats). hOCT1 was overexpressed in human hepatoma cells (HuH7 and HepG2). Sorafenib and regorafenib uptake was determined by HPLC-MS/MS. Key results hOCT1 overexpression enhanced sorafenib, but not regorafenib, quinine-inhibitable uptake by hepatoma cells. In rodent HCC, Oct1 was downregulated, which was accompanied by impaired sorafenib uptake. In mice with s.c.-implanted HCC, sorafenib inhibited the growth of hOCT1 overexpressing tumours. In human HCC, hOCT1 expression was inversely correlated with SLC22A1 promoter methylation, whereas demethylation with decitabine enhanced hOCT1 expression in hepatoma cells. Increased proportion of aberrant hOCT1 mRNA variants was found in HCC samples. In silico analysis identified six miRNAs as candidates to target hOCT1 mRNA. When overexpressed in HepG2 cells a significant hOCT1 mRNA decay was induced by hsa-miR-330 and hsa-miR-1468. Analysis of 39 paired tumour/adjacent samples from TCGA revealed that hsa-mir-330 was consistently upregulated in HCC. Conclusion and implications Impaired hOCT1 expression/function in HCC, in part due to epigenetic modifications, plays an important role in the poor pharmacological response of this cancer to sorafenib.

Background & Aims Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. Methods We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. Results Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. Conclusions We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. Lay summary In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases., Graphical abstract, Highlights • Hepatic MCJ levels were upregulated in patients with cholestatic liver disease. • MCJ absence resulted in decreased liver inflammation, neutrophil activation, and mitochondrial dysfunction after BDL. • MCJ deficiency protected the hepatocytes from ROS overproduction and ATP depletion. • In vivo inhibition of MCJ expression mitigated liver injury by bile acids. • Our results identified MCJ as a key regulator of cholestatic liver disease.

Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors,fosters this hypoxic pathway via phosphorylation of them RNA-binding protein HuR,a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses. Keywords: hypoxia; β-adrenergic signaling; breast cancer; mRNA regulation; nucleocytoplasmic shuttling; GRK2; HuR; HIF1α; VEGF &nbsp

Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from thephosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

Acute liver failure (ALF) is a life-threatening syndrome characterised by rapid hepatocellular necrosis due to various types of insults including drugs, autoimmune disorders or viral infections, and thus represents a disease with high mortality and elevated societal costs. At present, most ALF treatment strategies are rather aimed at simply preventing complications and decelerating disease progression. Unfortunately, the only curative treatment for ALF at present is liver transplantation, an option which is limited due to financial constraints, shortage of donor livers and immunosuppression-related complications.1 Therefore, novel therapeutic avenues for patients with ALF are urgently needed. Normally, mature hepatocytes can orchestrate liver regeneration on loss of liver mass. When severe liver insult occurs, however, hepatocytes are unable to differentiate and hepatic progenitor cells (HPCs) are activated. HPCs, embryonic stem cells (ES) and induced pluripotent stem cells (iPS) have been considered a potential source for cell replacement in the injured liver since they possess hepatic differentiation capacity.2 However, their clinical applicability remains still controversial. For long, it has been widely assumed that hepatocytes do not contribute directly to the progenitor or stem cell compartment. Recently, Chen and colleagues2 reported the dedifferentiation of progenitor cells from isolated mature hepatocytes, refuting the current dogma. Yet to be explored was the identification of molecular cascades that specifically dedifferentiate mature hepatocytes and activate a ‘fetal programme’. The Hippo/Yes-associated protein (YAP)-signalling pathway plays an essential role by determining cellular fates in the mammalian liver. Hippo signalling controls the phosphorylation of the transcriptional co-activator YAP whose constitutive activation in …

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73. Introduction, Supported by grants from the Department of Education of the Basque Government (PI2013-49 to JA and PI2012-42 to RB). JA is funded by the European Union (Grant Agreement number 602272). AMA and JLL’s work was supported by the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek Programs), the Innovation Technology Department of Bizkaia and the CIBERehd Network. The work of AC is supported by a Ramón y Cajal award, the Basque Department of Industry, Tourism and Trade (Etortek), ISCIII (PI13/00031), FERO VIII Fellowship, the BBVA foundation, MINECO (SAF2016-79381-R) and the European Research Council Starting Grant (336343). CIBERonc was co-funded with FEDER funds. AC-M was funded by a Juan de la Cierva program award and the European Union MSCA program (CIG 660191). RB was funded by MINECO grants BFU2011-25986 and BFU2014-52282-P and the Consolider Program (BFU2014-57703-REDC). FJB was funded by a MINECO grant (CTQ2014-56966-R). D.B. is funded by a MINECO FPI fellowship. We thank the MINECO for the Severo Ochoa Excellence accreditation (SEV-2016-0644).