BACKGROUND: Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs).
METHODS: Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes.
RESULTS: The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7.
CONCLUSIONS: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.

Dioxin-like molecules have been associated with endocrine disruption and liver disease. To better understand aryl hydrocarbon receptor (AHR) biology, metabolic phenotyping and liver proteomics were performed in mice following ligand-activation or whole-body genetic ablation of this receptor. Male wild type (WT) and Ahr -/- mice (Taconic) were fed a control diet and exposed to 3,3\',4,4\',5-pentachlorobiphenyl (PCB126) (61 nmol/kg by gavage) or vehicle for two weeks. PCB126 increased expression of canonical AHR targets (Cyp1a1 and Cyp1a2) in WT but not Ahr -/-. Knockouts had increased adiposity with decreased glucose tolerance; smaller livers with increased steatosis and perilipin-2; and paradoxically decreased blood lipids. PCB126 was associated with increased hepatic triglycerides in Ahr -/-. The liver proteome was impacted more so by Ahr -/- genotype than ligand-activation, but top gene ontology (GO) processes were similar. The PCB126-associated liver proteome was Ahr-dependent. Ahr principally regulated liver metabolism (e.g., lipids, xenobiotics, organic acids) and bioenergetics, but it also impacted liver endocrine response (e.g., the insulin receptor) and function, including the production of steroids, hepatokines, and pheromone binding proteins. These effects could have been indirectly mediated by interacting transcription factors or microRNAs. The biologic roles of the AHR and its ligands warrant more research in liver metabolic health and disease.

Rice is one of the most economically important commodities globally. However, rice plants are salt susceptible species in which high salinity can significantly constrain its productivity. Several physiological parameters in adaptation to salt stress have been observed, though changes in metabolic aspects remain to be elucidated. In this study, rice metabolic activities of salt-stressed flag leaf were systematically characterized. Transcriptomics and metabolomics data were combined to identify disturbed pathways, altered metabolites and metabolic hotspots within the rice metabolic network under salt stress condition. Besides, the feasible flux solutions in different context-specific metabolic networks were estimated and compared. Our findings highlighted metabolic reprogramming in primary metabolic pathways, cellular respiration, antioxidant biosynthetic pathways, and phytohormone biosynthetic pathways. Photosynthesis and hexose utilization were among the major disturbed pathways in the stressed flag leaf. Notably, the increased flux distribution of the photorespiratory pathway could contribute to cellular redox control. Predicted flux statuses in several pathways were consistent with the results from transcriptomics, end-point metabolomics, and physiological studies. Our study illustrated that the contextualized genome-scale model together with multi-omics analysis is a powerful approach to unravel the metabolic responses of rice to salinity stress.

Salvia miltiorrhiza Bunge is an herb rich in bioactive tanshinone and salvianolic acid compounds. It is primarily used as an effective medicine for treating cardiovascular and cerebrovascular diseases. Liposoluble tanshinones and water-soluble phenolic acids are a series of terpenoids and phenolic compounds, respectively. However, the regulation mechanism for the simultaneous promotion of tanshinone and salvianolic acid biosynthesis remains unclear. This study identified a R2R3-MYB subgroup 20 transcription factor (TF), SmMYB98, which was predominantly expressed in S. miltiorrhiza lateral roots. The accumulation of major bioactive metabolites, tanshinones, and salvianolic acids, was improved in SmMYB98 overexpression (OE) hairy root lines, but reduced in SmMYB98 knockout (KO) lines. The qRT-PCR analysis revealed that the transcriptional expression levels of tanshinone and salvianolic acid biosynthesis genes were upregulated by SmMYB98-OE and downregulated by SmMYB98-KO. Dual-Luciferase (Dual-LUC) assays demonstrated that SmMYB98 significantly activated the transcription of SmGGPPS1, SmPAL1, and SmRAS1. These results suggest that SmMYB98-OE can promote tanshinone and salvianolic acid production. The present findings illustrate the exploitation of R2R3-MYB in terpenoid and phenolic biosynthesis, as well as provide a feasible strategy for improving tanshinone and salvianolic acid contents by MYB proteins in S. miltiorrhiza.