Histone H3 clipping, a unique but evolutionarily conserved post-translational modification that irreversibly removes the N-terminal tail of H3, has been reported across diverse eukaryotic lineages. In Tetrahymena thermophila, a ciliate with nuclear dimorphism, H3 clipping is a bona fide proteolytic event generating H3^F (H3-Fast) by removing the first six N-terminal amino acids, specifically in the transcriptionally silent micronucleus (MIC). However, the detection of H3^F remains technically demanding, time-consuming, and lacks spatio-temporal resolution. To overcome this, a 2 × branched peptide antigen was developed to generate a high-specificity antibody that exclusively recognizes H3^F, effectively distinguishing it from full-length H3 and other truncation variants. This antibody eliminated the need for labor-intensive MIC isolation and histone extraction, enabling rapid, small-scale detection directly from whole-cell lysates. Using this antibody, dynamic subcellular localization of H3^F was investigated through different cell stages, revealing its persistence during vegetation, starvation and early conjugation. However, H3^F disappeared concurrently with macronuclear anlage formation, supporting the notation that removal of H3^F is a prerequisite for the new macronucleus development. Comparative analyses further revealed that H3 Ser10 phosphorylation, though previously used as an alternative H3^F marker, actually occurs strictly after clipping, refining the temporal hierarchy of these two chromatin events. This work provides the first in situ, high-resolution method to track endogenous H3 clipping, providing both a technical platform and new biological insight into the developmental regulation of proteolytic histone modifications.

In this study, we investigated the molecular recognition mechanisms of shark-derived single-domain antibodies (ssdAbs) targeting fluoroquinolones using an integrated approach that combines in silico homologous modeling, molecular dynamics simulations, molecular docking, and alanine scanning mutagenesis. Three ssdAbs—2E6, 1N9, and 1O17—specific to enrofloxacin, norfloxacin, and ofloxacin, respectively, were selected based on previous work. Through AlphaFold2 and GalaxyWEB, the protein structures of these ssdAbs were predicted and optimized, followed by molecular dynamics simulations to emulate realistic protein behavior in a solvent environment. Molecular docking, alanine scanning mutagenesis, and subsequent verifications identified 30N and 93W of 2E6; 30N, 89R, 98Y, and 99D of 1N9; 100W and 101R of 1O17, all located within the complementarity determining region 3 loop, as critical for antigen binding. These residues primarily interact with their targets through hydrogen bonds, salt bridges, π–π stackings, and cation–π interactions. This study revealed, for the first time, the binding mechanism of ssdAbs to fluoroquinolones from a theoretical perspective, emphasizing the importance of aromatic and polar residues in recognizing characteristic epitopes, such as the carboxyl group at the C3 position and the 1-piperazinyl group at the C7 position. Our findings provide valuable insights for the rational design and enhancement of ssdAbs for detecting small molecule hazards in aquaculture.