Oleic acid (OA), a monounsaturated fatty acid (MUFA), has previously been shown to reverse saturated fatty acid palmitic acid (PA)-induced hepatic insulin resistance (IR). However, its underlying molecular mechanism is unclear. In addition, previous studies have shown that eicosapentaenoic acid (EPA), a ω-3 polyunsaturated fatty acid (PUFA), reverses PA-induced muscle IR, but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes in HepG2 cells after treatment with different free fatty acids (FFAs). A total of 234 proteins were determined to be differentially expressed after PA+OA treatment. Their functions were mainly related to responses to stress and endogenous stimuli, lipid metabolic process, and protein binding. For PA+EPA treatment, the PA-induced expression changes of 1326 proteins could be reversed by EPA, 415 of which were mitochondrial proteins, with most of the functional proteins involved in oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species (ROS) play a role in OA- and EPA-reversed PA-induced IR, respectively. EPA and OA alleviated PA-induced abnormal adenosine triphosphate (ATP) production, ROS generation, and calcium (Ca2+) content. Importantly, H2O2-activated production of ROS increased the protein expression of JUN, further resulting in IR in HepG2 cells. Taken together, we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.

Polymerase-coupled nanopore sequencing requires DNA polymerases with strong strand displacement activity and high processivity to sustain continuous signal generation. In this study, we characterized two novel B family DNA polymerases, SRHS and BBum, isolated from Bacillus phages SRT01hs and BeachBum, respectively. Both enzymes exhibited robust strand displacement, 3′→5′ exonuclease activity, and maintained processivity under diverse reaction conditions, including across a broad temperature range (10–45 °C) and in the presence of multiple divalent metal cofactors (Mg²⁺, Mn²⁺, Fe²⁺), comparable to the well-characterized Phi29 polymerase. Through biochemical analysis of mutants designed using AlphaFold3-predicted structural models, we identified key residues (G96, M97, D486 in SRHS; S97, M98, A493 in BBum) that modulated exonuclease activity, substrate specificity and metal ion utilization. Engineered variants SRHS_F and BBum_Pro_L efficiently incorporated unnatural nucleotides in the presence of Mg²⁺—a function not observed in Phi29 and other wild-type strand-displacing B family polymerases. These combined biochemical features highlight SRHS and BBum as promising enzymatic scaffolds for nanopore-based long-read sequencing platforms.

Isothermal titration calorimetry (ITC) provides direct insight into the energetics of DNA polymerase function, including binding, catalysis, and exonuclease activity. We characterized a Phi29 mutant polymerase (SS_01) engineered to incorporate non-natural nucleotides in the presence of Mg²⁺, a function absent in the wild-type enzyme. ITC analyses revealed that SS_01 binding to the primed template was strongly influenced by metal ions. In the presence of Mg²⁺, the polymerase displayed tight binding (KD = 243 nM) and a clear exothermic signal, indicating activation of a large fraction of catalytically competent molecules. By contrast, in the presence of Ca²⁺, binding produced weaker exothermic signals (KD = 317 nM), suggesting less efficient binding complex formation. During dNTP- or oligonucleotide-tagged dNTP-driven polymerization, ITC profiles with Mg²⁺ exhibited pronounced endothermic heat changes, whereas with Ca²⁺, only minimal heat changes were observed. When binding only oligonucleotide-tagged dNTPs, the polymerases showed distinct thermodynamic behavior: in the presence of Mg²⁺, high substrate concentrations induced endothermic responses, while in the absence of catalytic ions, binding remained exothermic. Exonuclease activity monitored using unmodified oligonucleotides yielded strong exothermic signals in the presence of Mg²⁺ but weak responses in the presence of Ca²⁺, confirming strict ion dependence. Together, these data demonstrate that ITC directly captures the metal ion-dependent energetics of SS_01, providing mechanistic insight into its polymerization and exonuclease functions.