Freezing is used as a readout of fear memory in rodents, but it remains unclear how distinct freezing features reflect discrimination and generalization of threatening and neutral contexts. Using pose-tracking and unsupervised analysis in male and female mice undergoing contextual fear conditioning, we show that fear in neutral a context is expressed through fragmented freezing that resist experience-dependent updating, in contrast with more sustained, uninterrupted, and adaptable freezing in a threatening context. Neuronal recordings revealed that recalling neutral context fear is associated with increased activity of dentate gyrus somatostatin inhibitory neurons (SST INs). Chemogenetic suppression of SST INs during fear memory updating selectively facilitated neutral context freezing adaptation while activation prevented generalization of contextual information by restricting long-term fear reduction in contexts that differed from the updating context. These findings identify DG SST INs as context-specific modulators of fear memory updating and suggest that freezing dynamics serve as a behavioral signature of generalization.
NADH fluorescence lifetime imaging is a widely used, label-free way to visualize cell metabolism, typically read as a balance between glycolysis and oxidative respiration. This reading assumes that the effective NADH-binding landscape remains sufficiently stable during the experiment. This assumption is generally best protected during brief perturbations, but can fail in two ways: slowly, as prolonged treatments or differentiation reshape the identity and abundance of NADH-binding enzymes over hours to days; and rapidly, as metabolites can transiently alter their engagement with NADH even without changes in protein abundance. In this Perspective, we outline when each concern applies and offer practical guidance for experimental design and interpretation.
Fruit ripening is a complex developmental process involving coordinated changes in gene expression, metabolism, and epigenetic regulation. How DNA methylation dynamics influence gene transcription and metabolite accumulation during black raspberry (Rubus occidentalis) fruit ripening remains poorly understood. Here, we performed developmental-series transcriptome, methylome, and metabolome analysis; the results from this analysis reveal extensive epigenetic reprogramming during black raspberry fruit ripening. Fruit tissues exhibit globally higher DNA methylation than leaves, particularly in CHG and CHH contexts. Local methylation in all cytosine contexts progressively decreases in promoter regions during ripening, whereas CHG and CHH methylation increase in transposon-rich regions. Two primary methylation transitions—promoter hypomethylation and CHH hypermethylation—govern transcriptional shifts in genes involved in ripening processes. Methyl-binding transcription factors with activation potential likely promote CHH hypermethylation-linked transcriptional activation. Multi-omics integration reveals that coordinated anthocyanin accumulation parallels expression of biosynthetic and regulatory genes within coherent networks. Elevated non-CG methylation in heterochromatin coincides with increased transcription of histone variants mediating chromatin compaction, suggesting chromatin remodelers fine-tune accessibility for methyltransferases. Our study highlights both genome-wide and locus-specific epigenetic reprogramming and demonstrates a coordinated interplay between DNA methylation and transcriptional regulation during black raspberry fruit ripening.
Insulin resistance and pancreatic β-cell dysfunction are closely associated with impaired mitophagy, forming a core pathological mechanism in gestational diabetes mellitus (GDM). This study investigates the role of isofalcarintriol (IFT), a carrot-derived polyacetylene, in regulating mitochondrial function in GDM-related β-cells. Here, we use a high-fat diet-induced C57BL/6 J pregnant mouse model and high-glucose/palmitic acid (HG/PA)-treated MIN6 β-cells; IFT ameliorates hyperglycemia and insulin resistance, promotes β-cell maturation and proliferation, induces mitophagy, and enhances β-cell antioxidant capacity. Mechanistically, IFT promotes the dissociation of macrophage migration inhibitory factor (MIF) from ubiquitin-specific protease 7 (USP7) through its C224 residue, and induces MIF ubiquitination through F50/L59 residues, thereby driving the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1)/Parkin-mediated mitophagy and pancreatic injury recovery. A mitophagy inhibitor eliminates the beneficial effects of IFT, while PINK1 knockdown reverses the mitophagy-promoting effect induced by MIF knockdown. This study reveals IFT as a potent antioxidant protecting β-cells against GDM-induced injury through MIF, highlighting its potential value in GDM treatment.
V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an immune checkpoint that suppresses T-cell responses and has emerged as a promising target for cancer immunotherapy. Here we show, using atomistic molecular dynamics simulations of both truncated glycan-free and full-length glycosylated membrane-bound human VISTA, that the extracellular and transmembrane domains remain structurally stable, while the unusually long CC′ loop is stabilized by extensive hydrogen-bonding interactions. Principal component analysis and conformational clustering reveal a recurrent rotational motion of the extracellular domain relative to the membrane, producing two conformational states: an “Up” state with a solvent-exposed CC′ loop and a “Down” state in which the loop transiently associates with the membrane. These transitions are coupled to proline-mediated bending of the transmembrane helix. We propose that this membrane-coupled Up/Down switching regulates CC′ loop accessibility, providing a structural mechanism that may influence VISTA-mediated immune checkpoint signaling.
