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Abstract The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical l-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using l-cysteine and/or l-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

Abstract Background Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. Methods Cholangiocarcinogenesis was induced in rats (TAA) and mice (Jnk Δhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. Results Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. Conclusions In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

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.

BackgroundThough the precise criteria for accessing LT are consistently being applied, HCC recurrence (HCC-R_LT) still affects more than 15% of the patients. We analyzed the clinical, histopathological, and biological features of patients with HCC to identify the predictive factors associated with cancer recurrence and survival after LT.MethodsWe retrospectively analyzed 441 patients with HCC who underwent LT in our center. Overall, 70 (15.8%) of them developed HCC-R_LT. We matched them by age at transplant and etiology with 70 non-recurrent patients. A comparable cohort from the Liver Transplant Centre of Bologna served as validation. The clinical and biochemical characteristics and pre-LT criteria (Milan, Metroticket, Metroticket_AFP, and AFP model) were evaluated. Histological analysis and immunohistochemistry for angiopoietin-2 in the tumor and non-tumor tissue of explanted livers were performed. Patients’ follow-up was until death, last clinical evaluation, or 31 December 2021. In patients with HCC-R_LT, the date of diagnosis of recurrence and anatomical site has been reported; if a biopsy of recurrence was available, histologic and immunohistochemical analyses were also performed.ResultsPatients were followed up for a mean period of 62.7 ± 54.7 months (median, 39 months). A higher risk of HCC-R_LT was evident for factors related indirectly (AFP) or directly (endothelial angiopoietin-2, microvascular invasion) to biological HCC aggressiveness. In multivariate analysis, only angiopoietin-2 expression was independently associated with recurrence. Extremely high levels of endothelial angiopoietin-2 expression were also found in hepatic recurrence and all different metastatic locations. In univariate analysis, MELD, Metroticket_AFP Score, Edmondson–Steiner grade, microvascular invasion, and endothelial angiopoietin-2 were significantly related to survival. In multivariate analysis, angiopoietin-2 expression, Metroticket_AFP score, and MELD (in both training and validation cohorts) independently predicted mortality. In time-dependent area under receiver operating characteristic curve analysis, the endothelial angiopoietin-2 expression had the highest specificity and sensitivity for recurrence (AUC 0.922, 95% CI 0.876–0.962, p < 0.0001).ConclusionsEndothelial angiopoietin-2 expression is a powerful independent predictor of post-LT tumor recurrence and mortality, highlighting the fundamental role of tumor biology in defining the patients’ prognosis after liver transplantation. The great advantage of endothelial angiopoietin-2 is that it is evaluable in HCC biopsy before LT and could drive a patient’s priority on the waiting list.

Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.

Abstract Iron overload and cellular senescence have been implicated in liver fibrosis, but their possible mechanistic connection has not been explored. To address this, we have delved into the role of iron and senescence in an experimental model of chronic liver injury, analyzing whether an iron chelator would prevent liver fibrosis by decreasing hepatocyte senescence. The model of carbon tetrachloride (CCl4) in mice was used as an experimental model of liver fibrosis. Results demonstrated that during the progression of liver fibrosis, accumulation of iron occurs, concomitant with the appearance of fibrotic areas and cells undergoing senescence. Isolated parenchymal hepatocytes from CCl4-treated mice present a gene transcriptomic signature compatible with iron accumulation and senescence, which correlates with induction of Reactive Oxygen Species (ROS)-related genes, activation of the Transforming Growth Factor-beta (TGF-β) pathway and inhibition of oxidative metabolism. Analysis of the iron-related gene signature in a published single-cell RNA-seq dataset from CCl4-treated livers showed iron accumulation correlating with senescence in other non-parenchymal liver cells. Treatment with deferiprone, an iron chelator, attenuated iron accumulation, fibrosis and senescence, concomitant with relevant changes in the senescent-associated secretome (SASP), which switched toward a more anti-inflammatory profile of cytokines. In vitro experiments in human hepatocyte HH4 cells demonstrated that iron accumulates in response to a senescence-inducing reagent, doxorubicin, being deferiprone able to prevent senescence and SASP, attenuating growth arrest and cell death. However, deferiprone did not significantly affect senescence induced by two different agents (doxorubicin and deoxycholic acid) or activation markers in human hepatic stellate LX-2 cells. Transcriptomic data from patients with different etiologies demonstrated the relevance of iron accumulation in the progression of liver chronic damage and fibrosis, correlating with a SASP-related gene signature and pivotal hallmarks of fibrotic changes. Altogether, our study establishes iron accumulation as a clinically exploitable driver to attenuate pathological senescence in hepatocytes.

Background & Aims: Expression of P21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD); however, the underlying mechanisms remain unknown. In the present study, we investigated the function of CDKN1A in SLD. Methods: CDKN1A expression levels were evaluated in different patient cohorts with SLD, fibrosis, and advanced chronic liver disease (ACLD). Cdkn1a-/- and Cdkn1a+/+ mice were fed with either a Western diet (WD), a Lieber-DeCarli (LdC) diet plus multiple EtOH (ethanol) binges, or a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver). Primary hepatocytes were isolated and functional assays performed. Results: A significant increase in CDKN1A expression was observed in patients with steatohepatitis and fibrosis (with a positive correlation with both NAFLD Activity Score and fibrosis staging scores), cirrhosis and ACLD. Cdkn1a+/+ mice, fed a DuAL diet exhibited liver injury and cell death increased reactive oxygen species (ROS), and markers of senescence (γH2AX, β-GAL, Cdkn1a/p53) contributing to steatosis and inflammation. In contrast, Cdkn1a-/- mutant mice showed a significant decrease in senescence-associated markers as well as in markers of liver injury, hepatic steatosis and an increase in fatty acid oxidation and reduction in free fatty acid uptake as well as de novo lipogenesis. Mechanistically, activation of the AMPK-SIRT3 was observed in Cdkn1a-deleted animals. Conclusions: Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis via the AMPK-SIRT3 axis. CDKN1A expression was found to be directly correlated with increased severity of NAFLD Activity Score and fibrosis in patients with SLD. CDKN1A could be a potential theragnostic target for the treatment of metabolic dysregulation in patients with SLD, with and without alcohol consumption. Impact and implications:: Expression of p21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD), but the molecular mechanisms remain elusive. Interestingly, in this study we found that Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis, via the AMPK-SIRT3 axis. Translationally, Cdkn1a expression was found to be directly correlated with increased severity of NAFLD Activity Score (NAS) and fibrosis in SLD patients, and therefore, CDKN1A might be used potential theragnostic target for the treatment of metabolically induced SLD, with and without alcohol consumption.

Summary: The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.

Abstract Obesity is associated with adipose tissue dysfunction through the differentiation and expansion of pre-adipocytes to adipocytes (hyperplasia) and/or increases in size of pre-existing adipocytes (hypertrophy). A cascade of transcriptional events coordinates the differentiation of pre-adipocytes into fully differentiated adipocytes; the process of adipogenesis. Although nicotinamide N-methyltransferase (NNMT) has been associated with obesity, how NNMT is regulated during adipogenesis, and the underlying regulatory mechanisms, remain undefined. In present study we used genetic and pharmacological approaches to elucidate the molecular signals driving NNMT activation and its role during adipogenesis. Firstly, we demonstrated that during the early phase of adipocyte differentiation NNMT is transactivated by CCAAT/Enhancer Binding Protein beta (CEBPB) in response to glucocorticoid (GC) induction. We found that Nnmt knockout, using CRISPR/Cas9 approach, impaired terminal adipogenesis by influencing the timing of cellular commitment and cell cycle exit during mitotic clonal expansion, as demonstrated by cell cycle analysis and RNA sequencing experiments. Biochemical and computational methods showed that a novel small molecule, called CC-410, stably binds to and highly specifically inhibits NNMT. CC-410 was, therefore, used to modulate protein activity during pre-adipocyte differentiation stages, demonstrating that, in line with the genetic approach, chemical inhibition of NNMT at the early stages of adipogenesis impairs terminal differentiation by deregulating the GC network. These congruent results conclusively demonstrate that NNMT is a key component of the GC-CEBP axis during the early stages of adipogenesis and could be a potential therapeutic target for both early-onset obesity and glucocorticoid-induced obesity.

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.

Marine algae are valuable sources of bioactive compounds that have the potential to be used in the management of various pathologies. Despite the increasing prevalence of NAFLD, the absence of an approved effective pharmacological treatment with demonstrable effectiveness persists. In this context, the aim of the present study is to assess the effect of Gracilaria vermiculophylla red seaweed dietary supplementation on hepatic lipid accumulation, as well as on oxidative stress, inflammation and fibrosis- related markers on obese fa/fa Zucker rats fed with a standard diet, supplemented or not with 2.5% or 5% dehydrated Gracilaria vermiculophylla. After a six-week supplementation with the macroalga, no significant reduction in hepatic total lipid content or hepatic triglyceride content was observed. However, both doses were able to diminish hepatic NEFA concentration by reducing de novo lipogenesis and increasing mitochondrial biogenesis. Moreover, supplementation with the dose of 2.5% improved some oxidative stress and inflammation-related markers. Supplementation with the dose of 5% did not exert these clear beneficial effects. Thus, this study demonstrates that while Gracilaria vermiculophylla may not mitigate hepatic steatosis, it could exert protective effects on the liver by reducing NEFA content and enhancing oxidative stress and inflammation parameters.

The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Abstract Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1 f/f ) and hepatocyte-specific knockout Xbp1 mice (Xbp1 ∆hepa ) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1–2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1 ∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1 ∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.

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.

Chronic liver diseases include a broad group of hepatic disorders from different etiologies and with varying degrees of progression and severity. Among them, non-alcoholic fatty (NAFLD) and alcoholic (ALD) liver diseases are the most frequent forms of expression, caused by either metabolic alterations or chronic alcohol consumption. The liver is the main regulator of energy homeostasis and metabolism of potentially toxic compounds in the organism, thus hepatic disorders often promote the release of harmful substances. In this context, there is an existing interconnection between liver and brain, with the well-named brain-liver axis, in which liver pathologies lead to the promotion of neurodegenerative disorders. Alzheimer’s (AD) and Parkinson’s (PD) diseases are the most relevant neurological disorders worldwide. The present work highlights the relevance of the liver-related promotion of these disorders. Liver-related hyperammonemia has been related to the promotion of perturbations in nervous systems, whereas the production of ketone bodies under certain conditions may protect from developing them. The capacity of the liver of amyloid-β (Aβ) clearance is reduced under liver pathologies, contributing to the development of AD. These perturbations are even aggravated by the pro-inflammatory state that often accompanies liver diseases, leading to the named neuroinflammation. The current nourishment habits, named as Western diet (WD) and alterations in the bile acid (BA) profile, whose homeostasis is controlled by the liver, have been also related to both AD and PD, whereas the supplementation with certain compounds, has been demonstrated to alleviate the pathologies.

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.

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/−1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.

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.

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.

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common form of chronic liver disease. The histological spectrum of NAFLD ranges from simple steatosis to chronic inflammation and liver fibrosis during Non-Alcoholic Steatohepatitis (NASH). The current view is that innate immune mechanisms represent a key element in supporting hepatic inflammation in NASH. Natural Killer (NK) cells are lymphoid cells and a component of the innate immune system known to be involved in NASH progression. Increasing evidence has shed light on the differential function of circulating and tissue-resident NK cells, as well as on the relevance of metabolism and the microenvironment in regulating their activity. Here, we revisit the complex role of NK cells as regulators of NASH progression as well as potential therapeutic approaches based on their modulation.

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.

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.

Acetaminophen-induced liver injury is one of the most common causes of liver failure and has to be treated within hours of the overdose. Here Barbier-Torres et al. show that targeting MCJ, a mitochondrial negative regulator, even 24 h after the overdose protects liver from acetaminophen-induced damage.

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.

Targeting PML in acute promyelocytic leukaemia has changed the outcome of patients with this disease. Here, the authors demonstrated that PML is also a potential therapeutic target in breast cancer where it specifically regulates cancer initiating cells and tumour progression through the transcriptional regulation of SOX9.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for Hepatocellular carcinoma (HCC), but he transition from NAFLD to HCC is poorly understood. Feature selection algorithms in human and genetically modified mice NAFLD and HCC microarray data were applied to generate signatures of NAFLD progression and HCC differential survival. These signatures were used to study the pathogenesis of NAFLD derived HCC and explore which subtypes of cancers that can be investigated using mouse models. Our findings show that: (I) HNF4 is a common potential transcription factor mediating the transcription of NAFLD progression genes (II) mice HCC derived from NAFLD co-cluster with a less aggressive human HCC subtype of differential prognosis and mixed etiology (III) the HCC survival signature is able to correctly classify 95% of the samples and gives Fgf20 and Tgfb1i1 as the most robust genes for prediction (IV) the expression values of genes composing the signature in an independent human HCC dataset revealed different HCC subtypes showing differences in survival time by a Logrank test. In summary, we present marker signatures for NAFLD derived HCC molecular pathogenesis both at the gene and pathway level.