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Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n  = 133) and follow-up ( n  = 120), acute non-DILI at onset (NDO; n  = 63) and follow-up ( n  = 42), and healthy volunteers (HV; n  = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94–0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65–0.78), but further technical and clinical validation of these candidate biomarkers is needed. Diagnosis of rare, unpredictable, drug-induced liver injury (DILI) is a significant challenge for patients, clinicians, and drug development. Here, the authors discover, evaluate, and validate potential blood biomarkers to diagnose DILI and distinguish it from alternative causes of liver injury.

In contrast to traditional CRISPR–Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH + hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals. Intravenous delivery of an adenine base editor and a single-guide RNA for the Fah gene can correct an A>G splice-site mutation in an adult mouse model of tyrosinaemia.

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase ( FAH ) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach. Hereditary tyrosinemia type 1 (HT1) is an inborn error of metabolism caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Here, the authors show in an animal model that HT1 can be treated via in vivo portal vein administration of a lentiviral vector carrying the human FAH transgene.

Fusarium head blight (FHB), caused by Fusarium graminearum is a devastating disease that affects global wheat production. F. graminearum encodes many effector proteins; however, its virulence mechanisms are poorly understood. In this study, we identify a secretory e ffector c andidate (FgEC10) that is essential for the virulence of F. graminearum . FgEC10 interacts strongly with wheat fumarylacetoacetate hydrolase (TaFAH) and accelerates its degradation via the 26S proteasome pathway. In addition, we show that TaFAH interacts with proteasome 26S subunit, non-ATPases 12 (TaPSMD12) and that FgEC10 enhances the interaction between TaFAH and TaPSMD12. RNA silencing or overexpression of TaFAH in wheat plants shows that TaFAH positively regulates wheat FHB resistance. Overexpression of TaFAH promotes the expression of genes associated with disease resistance and the heading period. Metabolomic analysis reveals that overexpression of TaFAH increases the levels of several amino acids in wheat, and exogenous application of some of these amino acids show an increase in F. graminearum resistance in the wheat spike and seedling. Collectively, our study reveals a pathogenic mechanism and provides a valuable gene resource for improving FHB resistance and promoting heading in wheat. A Fusarium graminearum effector is found to target wheat fumarylacetoacetate hydrolase for 26S proteasomal degradation. The hydrolase enhances resistance to Fusarium head blight by regulating defense genes and amino acid metabolism, offering a genetic target for wheat improvement.

This study investigated the impact of overexpressing the mitochondrial enzyme Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) in human osteosarcoma epithelial cells (U2OS) in vitro. While the downregulation or knockdown of FAHD1 has been extensively researched in various cell types, this study aimed to pioneer the exploration of how increased catalytic activity of human FAHD1 isoform 1 (hFAHD1.1) affects human cell metabolism. Our hypothesis posited that elevation in FAHD1 activity would lead to depletion of mitochondrial oxaloacetate levels. This depletion could potentially result in a decrease in the flux of the tricarboxylic acid (TCA) cycle, thereby accompanied by reduced ROS production. In addition to hFAHD1.1 overexpression, stable U2OS cell lines were established overexpressing a catalytically enhanced variant (T192S) and a loss-of-function variant (K123A) of hFAHD1. It is noteworthy that homologs of the T192S variant are present in animals exhibiting increased resistance to oxidative stress and cancer. Our findings demonstrate that heightened activity of the mitochondrial enzyme FAHD1 decreases cellular ROS levels in U2OS cells. However, these results also prompt a series of intriguing questions regarding the potential role of FAHD1 in mitochondrial metabolism and cellular development.

A quarter of humanity is estimated to have been exposed to Mycobacterium tuberculosis ( Mtb ) with a 5–10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n = 63) or did not progress to TB (controls, n = 63). Transcriptomic profiling of monocyte-derived DCs and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Four genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH , the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb -infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.

Normal cells become cancer cells after a malignant transformation, but whether cancer cells can be reversed to normal status remains elusive. Here, we report that the combination of hepatocyte nuclear factor 1A (HNF1A), HNF4A and forkhead box protein A3 (FOXA3) synergistically reprograms hepatocellular carcinoma (HCC) cells to hepatocyte-like cells (reprogrammed hepatocytes, rHeps). Our results show that rHeps lose the malignant phenotypes of cancer cells and retrieve hepatocyte-specific characteristics including hepatocyte-like morphology; global expression pattern of genes and specific biomarkers of hepatocytes; and the unique hepatic functions of albumin (ALB) secretion, glycogen synthesis, low-density lipoprotein (LDL) uptake, urea production, cytochrome P450 enzymes induction and drug metabolism. Intratumoral injection of these three factors efficiently shrank patient-derived tumor xenografts and reprogrammed HCC cells in vivo. Most importantly, transplantation of rHeps in the liver of fumarylacetoacetate hydrolase-deficient (Fah−/−) mice led to the reconstruction of hepatic lobules and the restoration of hepatic function. Mechanistically, exogenous expression of HNF1A, HNF4A and FOXA3 in HCC cells initiated the endogenous expression of numerous hepatocyte nuclear factors, which promoted the conversion of HCC cells to hepatocyte-like cells. Collectively, our results indicate the successful conversion of hepatoma cells to hepatocyte-like cells, not only extending our current knowledge of cell reprogramming but also providing a route towards a novel therapeutic strategy for cancer.

Dexamethasone (Dex) is a synthetic glucocorticoid that has anti-inflammatory and immunosuppressant effects and is used in several conditions such as asthma and severe allergy. Patients receiving Dex, either at a high dose or for a long time, might develop several side effects such as hyperglycemia, weight change, or osteoporosis due to its non-selectivity. Herein, we used liquid chromatography-tandem mass spectrometry-based comprehensive targeted metabolomic profiling as well as radiographic imaging techniques to study the side effects of Dex treatment in rats. The Dex-treated rats suffered from a ∼20% reduction in weight gain, hyperglycemia (145 mg/dL), changes in serum lipids, and reduction in total serum alkaline phosphatase (ALP) (∼600 IU/L). Also, compared to controls, Dex-treated rats showed a distinctive metabolomics profile. In particular, serum amino acids metabolism showed six-fold reduction in phenylalanine, lysine, and arginine levels and upregulation of tyrosine and hydroxyproline reflecting perturbations in gluconeogenesis and protein catabolism which together lead to weight loss and abnormal bone metabolism. Sorbitol level was markedly elevated secondary to hyperglycemia and reflecting activation of the polyol metabolism pathway causing a decrease in the availability of reducing molecules (glutathione, NADPH, NAD ). Overexpression of succinylacetone (4,6-dioxoheptanoic acid) suggests a novel inhibitory effect of Dex on hepatic fumarylacetoacetate hydrolase. The acylcarnitines, mainly the very long chain species (C12, C14:1, C18:1) were significantly increased after Dex treatment which reflects degradation of the adipose tissue. In conclusion, long-term Dex therapy in rats is associated with a distinctive metabolic profile which correlates with its side effects. Therefore, metabolomics based profiling may predict Dex treatment-related side effects and may offer possible novel therapeutic interventions.

BackgroundDiagnosis of drug-induced liver injury (DILI) remains a significant challenge in interventional clinical trials during drug development and in clinical practice. We aimed to discover novel diagnostic biomarkers using proteomics and to evaluate their performance characteristics in distinguishing DILI from acute and chronic liver injury from other etiologies.MethodsAdults with idiosyncratic DILI and acute non-DILI liver disease controls were recruited to the Prospective European DILI Registry. Case definitions and causality assessment were performed according to European Association for the Study of the Liver recommendations. Tandem Mass Tag (TMT) labelled quantitative proteomics was performed in serum samples from a discovery set of healthy volunteers (HV, n=10), patients with DILI at onset (DO) and recovery (DR) (n=10 each); acute non-DILI controls at onset (NDO) and recovery (NDR) (n=5 each) and patients with non-alcoholic fatty liver disease (n=10). The performance of top biomarker candidates was assessed in a confirmatory cohort with HV (n=60), DO (n=83), DR (n=85), NDO (n=35) and NDR (n=21) using peptide-based targeted mass spectrometry. Novel biomarkers and patient status were evaluated using univariate and multivariate models.ResultsGlobal proteomic profiling of the discovery cohort revealed 2323 proteins, of which 12 novel biomarkers were chosen based on differential expression, liver specificity, and mechanistic relevance to liver biology/pathogenesis. In the confirmatory analysis, serum concentration of the novel biomarkers was significantly different at DO and returned to levels comparable to HV during recovery (DR). When the diagnostic performance of these 12 biomarkers was assessed individually, along with the promising biomarker Cytokeratin 18 (CK18, Area Under the Receiver Operator Characteristic (AUROC) Curve = 0.96), the following biomarkers demonstrated the highest sensitivity/specificity (DILI vs HV): Cytoplasmic aconitate hydratase (ACO1, 0.99), Argininosuccinate synthase (ASS1, 0.98), Fumarylacetoacetase (FAH, 0.98), Carbamoyl-phosphate synthase (CPS1, 0.96), Fructose-bisphosphate aldolase B (ALDOB, 0.94), 4-Hydroxyphenylpyruvate dioxygenase (HPD, 0.94), Ornithine carbamoyltransferase (OTC, 0.92).ConclusionA TMT-based quantitative proteomic profiling approach identified novel putative biomarkers, which are candidates for further evaluation of their role in the diagnosis and prognostication of DILI.

Hereditary tyrosinemia type I (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (Fah). Fah-deficient mice and pigs are phenotypically analogous to human HT1, but do not recapitulate all the chronic features of the human disorder, especially liver fibrosis and cirrhosis. Rats as an important model organism for biomedical research have many advantages over other animal models. Genome engineering in rats is limited till the availability of new gene editing technologies. Using the recently developed CRISPR/Cas9 technique, we generated Fah −/− rats. The Fah −/− rats faithfully represented major phenotypic and biochemical manifestations of human HT1, including hypertyrosinemia, liver failure and renal tubular damage. More importantly, the Fah −/− rats developed remarkable liver fibrosis and cirrhosis, which have not been observed in Fah mutant mice or pigs. Transplantation of wild-type hepatocytes rescued the Fah −/− rats from impending death. Moreover, the highly efficient repopulation of hepatocytes in Fah −/− livers prevented the progression of liver fibrosis to cirrhosis and in turn restored liver architecture. These results indicate that Fah −/− rats may be used as an animal model of HT1 with liver cirrhosis. Furthermore, Fah −/− rats may be used as a tool in studying hepatocyte transplantation and a bioreactor for the expansion of hepatocytes.