RESUMEN
We designed 6-dimethylamino 3-methyleneisoindolin-1-one as an environment-sensitive fluorophore, examining its applications for protein labeling. Synthesized 3-methyleneisoindolin-1-one exhibits solvatochromic fluorescence (λemmax; 472 nm in 2-PrOH, 512 nm in H2O). A positive linear dependence between λemmax and solvent dielectric constant (DC), as well as between Stokes shift and DC, and a negative correlation between fluorescence quantum yield and DC are observed in protic solvents. These properties are similar to those of the oxygen isosteric fluorophore, 4-dimethylaminophthalimide, a slovatochromic fluorophore utilized for labeling oligodeoxynucleotides (ODNs) and peptides. Notably, fluorescence intensity of 3-methyleneisoindolin-1-one is higher than the phthalimide in protic solvents used in this study. The 3-methyleneisoindolin-1-one demonstrated the higher stability in pH 8 solution than in pH 6 solution in contrast to the stability profile of the phthalimide, which was stable at pH 6 but was hydrolyzed at pH 8. We also synthesized an o-keto benzaldehyde derivative that converts a primary amine to 6-dimethylamino 3-methyleneisoindolin-1-one under biocompatible conditions and introduced it into ODNs for turn-on fluorescent protein labeling. The synthesized ODN with a protein-binding sequence of Escherichia coli DnaA was employed to modify the DNA-binding domain of DnaA, and the fluorescent properties of the modified protein were investigated.
RESUMEN
In middle to late 2023, a sublineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. We performed multiscale investigations, including phylogenetic analysis, epidemic dynamics modeling, infection experiments using pseudoviruses, clinical isolates, and recombinant viruses in cell cultures and experimental animals, and the use of human sera and antiviral compounds, to reveal the virological features of the newly emerging EG.5.1 variant. Our phylogenetic analysis and epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T are critical to its increased viral fitness. Experimental investigations on the growth kinetics, sensitivity to clinically available antivirals, fusogenicity, and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 are comparable to those of XBB.1.5. However, cryo-electron microscopy revealed structural differences between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible in our experimental setup. Our multiscale investigations provide knowledge for understanding the evolutionary traits of newly emerging pathogenic viruses, including EG.5.1, in the human population.
RESUMEN
Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5.