Species Identification and Mycotoxigenic Potential of Aspergillus Section Flavi Isolated from Maize Marketed in the Metropolitan Region of Asunción, Paraguay.

Zea mays var. amylacea and Zea mays var. indurata are maize ecotypes from Paraguay. Aspergillus section Flavi is the main spoilage fungus of maize under storage conditions. Due to its large intraspecific genetic variability, the accurate identification of this fungal taxonomic group is difficult. In the present study, potential mycotoxigenic strains of Aspergillus section Flavi isolated from Z. mays var. indurata and Z. mays var. amylacea that are marketed in the metropolitan region of Asunción were identified by a polyphasic approach. Based on morphological characters, 211 isolates were confirmed to belong to Aspergillus section Flavi. A subset of 92 strains was identified as Aspergillus flavus by mass spectrometry MALDI-TOF and the strains were classified by MALDI-TOF MS into chemotypes based on their aflatoxins and cyclopiazonic acid production. According to the partial sequencing of ITS and CaM genes, a representative subset of 38 A. flavus strains was confirmed. Overall, 75 A. flavus strains (86%) were characterized as producers of aflatoxins. The co-occurrence of at least two mycotoxins (AF/ZEA, FUM/ZEA, and AF/ZEA/FUM) was detected for five of the Z. mays samples (63%). Considering the high mycological bioburden and mycotoxin contamination, maize marketed in the metropolitan region of Asunción constitutes a potential risk to food safety and public health and requires control measures.

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis.

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

Application of a gut-liver-on-a-chip device and mechanistic modelling to the quantitative in vitro pharmacokinetic study of mycophenolate mofetil.

Microphysiological systems (MPS) consisting of multiple linked organ-on-a-chip (OoC) components are highly promising tools with potential to provide more relevant in vitro to in vivo translation of drug disposition, efficacy and toxicity. A gut-liver OoC system was employed with Caco2 cells in co-culture with HT29 cells in the intestinal compartment and single donor primary hepatocytes in the hepatic compartment for the investigation of intestinal permeability, metabolism (intestinal and hepatic) and potential interplay of those processes. The prodrug mycophenolate mofetil was tested for quantitative evaluation of the gut-liver OoC due to the contribution of both gut and liver in its metabolism. Conversion of mycophenolate mofetil to active drug mycophenolic acid and further metabolism to a glucuronide metabolite was assessed over time in the gut apical, gut basolateral and liver compartments. Mechanistic modelling of experimental data was performed to estimate clearance and permeability parameters for the prodrug, active drug and glucuronide metabolite. Integration of gut-liver OoC data with in silico modelling allowed investigation of the complex combination of intestinal and hepatic processes, which is not possible with standard single tissue in vitro systems. A comprehensive evaluation of the mechanistic model, including structural model and parameter identifiability and global sensitivity analysis, enabled a robust experimental design and estimation of in vitro pharmacokinetic parameters. We propose that similar methodologies may be applied to other multi-organ microphysiological systems used for drug metabolism studies or wherever quantitative knowledge of changing drug concentration with time enables better understanding of biological effect.

Identification of an FXR-modulated liver-intestine hybrid state in iPSC-derived hepatocyte-like cells.

BACKGROUND & AIMS: Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLC) have enormous potential as a replacement for primary hepatocytes in drug screening, toxicology and cell replacement therapy, but their genome-wide expression patterns differ strongly from primary human hepatocytes (PHH). METHODS: We differentiated human induced pluripotent stem cells (hiPSC) via definitive endoderm to HLC and characterized the cells by single-cell and bulk RNA-seq, with complementary epigenetic analyses. We then compared HLC to PHH and publicly available data on human fetal hepatocytes (FH) ex vivo; we performed bioinformatics-guided interventions to improve HLC differentiation via lentiviral transduction of the nuclear receptor FXR and agonist exposure. RESULTS: Single-cell RNA-seq revealed that transcriptomes of individual HLC display a hybrid state, where hepatocyte-associated genes are expressed in concert with genes that are not expressed in PHH - mostly intestinal genes - within the same cell. Bulk-level overrepresentation analysis, as well as regulon analysis at the single-cell level, identified sets of regulatory factors discriminating HLC, FH, and PHH, hinting at a central role for the nuclear receptor FXR in the functional maturation of HLC. Combined FXR expression plus agonist exposure enhanced the expression of hepatocyte-associated genes and increased the ability of bile canalicular secretion as well as lipid droplet formation, thereby increasing HLCs' similarity to PHH. The undesired non-liver gene expression was reproducibly decreased, although only by a moderate degree. CONCLUSION: In contrast to physiological hepatocyte precursor cells and mature hepatocytes, HLC co-express liver and hybrid genes in the same cell. Targeted modification of the FXR gene regulatory network improves their differentiation by suppressing intestinal traits whilst inducing hepatocyte features. LAY SUMMARY: Generation of human hepatocytes from stem cells represents an active research field but its success is hampered by the fact that the stem cell-derived 'hepatocytes' still show major differences to hepatocytes obtained from a liver. Here, we identified an important reason for the difference, specifically that the stem cell-derived 'hepatocyte' represents a hybrid cell with features of hepatocytes and intestinal cells. We show that a specific protein (FXR) suppresses intestinal and induces liver features, thus bringing the stem cell-derived cells closer to hepatocytes derived from human livers.

Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies.

Microphysiological systems (MPS) are complex and more physiologically realistic cellular in vitro tools that aim to provide more relevant human in vitro data for quantitative prediction of clinical pharmacokinetics while also reducing the need for animal testing. The PhysioMimix liver-on-a-chip integrates medium flow with hepatocyte culture and has the potential to be adopted for in vitro studies investigating the hepatic disposition characteristics of drug candidates. The current study focusses on liver-on-a-chip system exploration for multiple drug metabolism applications. Characterization of cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT) and aldehyde oxidase (AO) activities was performed using 15 drugs and in vitro to in vivo extrapolation (IVIVE) was assessed for 12 of them. Next, the utility of the liver-on-a-chip for estimation of the fraction metabolized (fm) via specific biotransformation pathways of quinidine and diclofenac was established. Finally, the metabolite identification opportunities were also explored using efavirenz as an example drug with complex primary and secondary metabolism involving a combination of CYP, UGT and sulfotransferase enzymes. A key aspect of these investigations was the application of mathematical modelling for improved parameter calculation. Such approaches will be required for quantitative assessment of metabolism and/or transporter processes in systems where medium flow and system compartments result in non-homogeneous drug concentrations. In particular, modelling was used to explore the effect of evaporation from the medium and it was found that the intrinsic clearance (CLint) might be underestimated by up to 40% for low clearance compounds if evaporation is not accounted for. Modelling of liver-on-a-chip in vitro data also enhanced the approach to fm estimation allowing objective assessment of metabolism models of different complexity. The resultant diclofenac fm,UGT of 0.64 was highly comparable with values reported previously in the literature. The current study demonstrates the integration of mathematical modelling with experimental liver-on-a-chip studies and illustrates how this approach supports generation of high quality of data from complex in vitro cellular systems.

Transcriptomic Cross-Species Analysis of Chronic Liver Disease Reveals Consistent Regulation Between Humans and Mice.

Mouse models are frequently used to study chronic liver diseases (CLDs). To assess their translational relevance, we quantified the similarity of commonly used mouse models to human CLDs based on transcriptome data. Gene-expression data from 372 patients were compared with data from acute and chronic mouse models consisting of 227 mice, and additionally to nine published gene sets of chronic mouse models. Genes consistently altered in humans and mice were mapped to liver cell types based on single-cell RNA-sequencing data and validated by immunostaining. Considering the top differentially expressed genes, the similarity between humans and mice varied among the mouse models and depended on the period of damage induction. The highest recall (0.4) and precision (0.33) were observed for the model with 12-months damage induction by CCl4 and by a Western diet, respectively. Genes consistently up-regulated between the chronic CCl4 model and human CLDs were enriched in inflammatory and developmental processes, and mostly mapped to cholangiocytes, macrophages, and endothelial and mesenchymal cells. Down-regulated genes were enriched in metabolic processes and mapped to hepatocytes. Immunostaining confirmed the regulation of selected genes and their cell type specificity. Genes that were up-regulated in both acute and chronic models showed higher recall and precision with respect to human CLDs than exclusively acute or chronic genes. Conclusion: Similarly regulated genes in human and mouse CLDs were identified. Despite major interspecies differences, mouse models detected 40% of the genes significantly altered in human CLD. The translational relevance of individual genes can be assessed at https://saezlab.shinyapps.io/liverdiseaseatlas/.

Chemical Analysis and In Vitro Bioactivity of Essential Oil of Laurelia sempervirens and Safrole Derivatives against Oomycete Fish Pathogens.

In this study, the essential oil (EO) from Laurelia sempervirens was analyzed by GC/MS and safrole (1) was identified as the major metabolite 1, was subjected to direct reactions on the oxygenated groups in the aromatic ring and in the side chain, and eight compounds (4 to 12) were obtained by the process. EO and compounds 4-12 were subjected to biological assays on 24 strains of the genus Saprolegnia, specifically of the species 12 S. parasitica and 12 S. australis. EO showed a significant effect against Saprolegnia strains. Compound 6 presents the highest activity against two resistant strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 25 to 100 and 75 to 125 µg/mL, respectively. The results show that compound 6 exhibited superior activities compared to the commercial controls bronopol and azoxystrobin used to combat these pathogens.

Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

Key Characteristics of Human Hepatotoxicants as a Basis for Identification and Characterization of the Causes of Liver Toxicity.

Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on preclinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize, and use mechanistic data when identifying hepatotoxicants.

Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae / Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae

Resumen Los hongos dematiáceos son un grupo heterogéneo de microorganismos capaces de sintetizar melanina. Las infecciones de este grupo que producen hifas en tejidos se denominan feohifomicosis y generalmente afectan la piel y tejidos vecinos. Presentamos el caso de un varón de 86 años con un tumor quístico blando progresivo en su mano y muñeca derecha, no asociado a dolor o signos inflamatorios. Se demostró una tenosinovitis de los flexores con pseudocapsula y sinovitis adherida a los tendones. El cultivo demostró un hongo dematiáceo compatible con Pleurostomophora richardsiae que se confirmó por secuenciación de la región ITS. La biopsia mostró una inflamación crónica granulomatosa e hifas. Después del drenaje quirúrgico, el paciente fue dado de alta sin terapia antifúngica, pero falleció por causas no relacionadas, tres meses después. Esta es la primera descripción de P. richardsiae como causa de feohifomicosis en Chile. Esta patología se puede sospechar cuando una lesión quística cutánea crónica involucra extremidades sin signos inflamatorios. Puede afectar a pacientes inmunocompetentes o inmunocomprometidos. El tratamiento contempla la escisión quirúrgica con o sin terapia antifúngica.

Abstract Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

Sonochemical Synthesis of 2'-Hydroxy-Chalcone Derivatives with Potential Anti-Oomycete Activity.

This work reports on the synthesis of eight new 2'-hydroxy-chalcones with potential anti-phytopathogenic applications in agroindustry, among others, via Claisen-Schmidt condensation and ultrasound assisted reaction. Assays showed three chalcones with allyl moieties strongly inhibited growth of phytopathogenic oomycete Phytophthora infestans; moreover, compound 8a had a half maximal effective concentration (EC50) value (32.5 µg/mL) similar to that of metalaxyl (28.6 µg/mL). A software-aided quantitative structure-activity relationship (QSAR) analysis of the whole series suggests that the structural features of these new chalcones-namely, the fluoride, hydroxyl, and amine groups over the carbon 3' of the chalcone skeleton-increase anti-oomycete activity.

Synthesis and Anti-Saprolegnia Activity of New 2',4'-Dihydroxydihydrochalcone Derivatives.

In the present study, seven 2',4'-dihydroxydihydrochalcone derivatives (compounds 3-9) were synthesized and their capacity as anti-Saprolegnia agents were evaluated against Saprolegnia parasitica, S. australis, S. diclina. Derivative 9 showed the best activity against the different strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values between 100-175 µg/mL and 100-200 µg/mL, respectively, compared with bronopol and fluconazole as positive controls. In addition, compound 9 caused damage and disintegration cell membrane of all Saprolegnia strains over the action of commercial controls.

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects.

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERKT188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERKT188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERKT188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERKT188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.

Inflammation-associated suppression of metabolic gene networks in acute and chronic liver disease.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.

[Hand cold abscess by a dematiaceous fungus. First report in Chile of Pleurostomophora richardsiae]. / Absceso frío en una mano por un hongo dematiáceo. Primera comunicación en Chile de Pleurostomophora richardsiae.

Dematiaceous fungi are a heterogeneous group of microorganisms able to synthesize melanin. Infections by this group that provoke tissular hyphae are called phaeohyphomycosis and usually involve skin and neighbor tissues. We present the case of a 86 years old men with a progressive soft cystic tumor in his right hand and wrist not associated to pain or inflammatory signs. A surgical intervention demonstrated flexor tenosynovitis with serous secretion, pseudocapsule and synovitis. Fungal culture demonstrated a dematiaceous fungi compatible with Pleurostomophora richardsiae that was confirmed by sequencing of the ITS region. Biopsy showed chronic inflammation with granuloma and hyphae. After surgical drainage, the patient was discharged without antifungal therapy but died of unrelated causes three month later. This is the first description of P. richardsiae as a cause of phaeohyphomycosis in Chile, a country with a template climate. Phaeohyphomycosis can be suspected when a chronic skin cystic lesion involves extremities without inflammatory signs, sometimes with an associated fistula. It may affect immunocompetent or immunosuppressed patients. Treatment involves surgical excision with or without antifungal therapy and prognosis is favorable.

Carveoylphenols and Their Antifungal Potential against Pathogenic Yeasts.

Candida is a genus of yeasts and is the most common cause of fungal infections worldwide. However, only a few antifungal drugs are currently available for the treatment of Candida infections. In the last decade, terpenophenols have attracted much attention because they often possess a variety of biological activities. In the search for new antifungals, eight carveoylphenols were synthesized and characterized by spectroscopic analysis. By using the broth microdilution assay, the compounds were evaluated for antifungal activities in vitro against four human pathogenic yeast, and structure-activity relationships (SAR) were derived. Noteworthy, in this preliminary study, compounds 5 and 6, have shown a significant reduction in the growth of all Candida strains tested. Starting from these preliminary results, we have designed the second generation of analogous in this class, and further studies are in progress in our laboratories.

Isolation and identification of compounds from the resinous exudate of Escallonia illinita Presl. and their anti-oomycete activity.

The resinous exudates from Escallonia illinita by products was characterized by FT-IR, NMR and HRMS. Six compounds were isolated and identified as follows: 1,5-diphenylpent-1-en-3-one (1), 4-(5-hydroxy-3,7-dimethoxy-4-oxo-4H-chromen-2-yl)phenyl acetate (2), pinocembrin (3), kaempferol 3-O-methylether (4), (3S,5S)-(E)-1,7-diphenylhept-1-ene-3,5-diol (5) and the new diarylheptanoid (3S,5S)-(E)-5-hydroxy-1,7-diphenylhept-1-en-3-yl acetate (6). The anti-oomycete potential of the resinous exudate, as well as the main compounds, was tested in vitro against Saprolegnia parasitica and Saprolegnia australis. The resinous exudate showed a strong anti-oomycete activity. In addition, the compounds 6, 1 and 3 demonstrated significant inhibition of Saprolegnia strains development. These findings strongly suggest that E. illinita is a potential biomass that could be used as a natural anti-oomycete product.

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation.

During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4α is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4α's Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4α during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation.