Gas hydrates have an important role in environmental and astrochemistry, as well as in energy materials research. Although it is widely accepted that gas accumulation is an important and necessary process during hydrate nucleation, how guest molecules aggregate remains largely unknown. Here, we have performed molecular dynamics simulations to clarify the nucleation path of methane hydrate. We demonstrated that methane gather with a three-body aggregate pattern corresponding to the free energy minimum of three-methane hydrophobic interaction. Methane molecules fluctuate around one methane which later becomes the central gas molecule, and when several methanes move into the region within 0.8 nm of the potential central methane, they act as directional methane molecules. Two neighbor directional methanes and the potential central methane form a three-body aggregate as a regular triangle with a distance of ~6.7 Å which is well within the range of typical methane-methane distances in hydrates or in solution. We further showed that hydrate nucleation and growth is inextricably linked to three-body aggregates. By forming one, two, and three three-body aggregates, the possibility of hydrate nucleation at the aggregate increases from 3/6, 5/6 to 6/6. The results show three-body aggregation of guest molecules is a key step in gas hydrate formation.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in clinical practice. This study examined the effects of mulberry leaf aqueous extract (MLQE) on lipid metabolism and gut microbiota in mice with high-fat diet-induced NAFLD. Using network pharmacology, we identified key targets of mulberry leaf and stem’s constituents for NAFLD regulation and validated their ameliorative effects through animal experiments. Our results demonstrated that interventions with orlistat (HFO), swimming exercise (HFS), and MLQE (HFM) significantly reduced weight gain, lipid accumulation, and oxidative stress in mace compared to the high-fat control (HFC) group. Histopathological analysis revealed improvements in liver steatosis, hepatocyte arrangement, inflammation, and adipocyte deformation. Molecular analyses indicated decreased mRNA levels of lipid synthesis genes (sterol regulatory element-binding protein 1 (SREBP1), bile salt export pump (BSEP), small heterodimer partner (SHP), and stearoyl-CoA desaturase-1 (SCD1)) and increased protein levels of farnesoid X receptor (FXR) and SHP in the intervention groups, consistent with RNA-seq findings. Additionally, RNA-seq analysis showed upregulation of fibroblast growth factor 21 (FGF21) and suppressor of cytokine signaling 2 (SOCS2), aligning with protein level observations. The interventions also modulated gut microbiota composition, reducing the Firmicutes-to-Bacteroidetes (F/B) ratio and adjusting the abundance of Acetatifactor, Roseburia, and Parasutterella. These findings suggest MLQE as a promising therapeutic approach for NAFLD, with lipid-lowering, antioxidative, and gut microbiota-regulating effects. FGF21 and SOCS2 may be key targets in NAFLD treatment.