International Journal of Molecular Sciences

Chemical modification is a valuable strategy for tuning enzyme functionality by introducing new reactive groups without disrupting the overall fold. Conventional amination using ethylenediamine (EDA) is effective, but the resulting modified proteins show limited reactivity for conjugation at neutral pH, and the modifier itself poses safety concerns due to its volatility and corrosive nature. Dihydrazides, in contrast, offer a safer and more versatile alternative: they operate through the same carboxyl-activation mechanism while enabling systematic investigation of chain-length effects. In this study, Thermomyces lanuginosus lipase (TLL) and Myceliophthora thermophila laccase (MTL) were modified using dihydrazides with different alkyl chain lengths (carbonyl (CZ), oxalyl (OX), succinyl (SC), and adipic (AA)), and compared to EDA-modified and unmodified enzymes to evaluate their effects on catalytic performance. Hydrazide-modified variants exhibited enhanced catalytic performance, reaching up to 2.5-fold (TLL-CZ) and 4.2-fold (MTL-AA and MTL-OX) higher efficiencies than unmodified and EDA-modified enzymes. Notably, AA provided the most consistent improvement across both enzymes (1.3-fold in TLL and the best in MTL). Molecular dynamics and docking analyses supported these findings, linking increased flexibility (higher RoG and RMSF) with higher k_cat, and changes in substrate binding with lower k_m. Overall, hydrazide-based modification broadens the spectrum of enzyme variants attainable through amination, while offering safer procedures, thus representing an alternative that overcomes the limitations of using EDA as a conventional aminating agent.

Open posterior lumbar surgery involves detaching paraspinal muscles from the spine to decompress neural tissues and to place instruments. While this operation improves the quality of life, it often has adverse effects on skeletal muscles like inflammation, degeneration, and fibrosis. Corticosteroids are well known for their anti-inflammatory function. In this study, we assessed the protective effects of intramuscular injection of corticosteroid on injured paraspinal muscles following surgery on the spine. C2C12 cells were co-exposed to hypoxia and lipopolysaccharide (LPS) to simulate ischemia and inflammatory response after muscle retraction to assess the effect of steroid. In vivo experiment, animals first underwent paraspinous muscle splitting with retractors to induce muscle injury, and later were assessed for neurobehavior, electrophysiology, and protein level related to inflammatory or regeneration following intramuscular (IM) steroid injection. Steroid rescued reduced cell viability caused by hypoxia + LPS, and attenuated induced protein expression of iNOS, COX2, Bad, and Bax. In neurobehavioral assessments (CatWalk, Ethovision, Von Frey test, and open field locomotor), retraction of paraspinous muscles worsened behaviors that were improved by IM steroid injections. The electrophysiology study showed that IM steroid injection lessened the muscle denervation caused by retraction. Similarly, IM steroid injections also attenuated dorsal root ganglion antigenicity of CGRP, Iba-1, and CD68 induced by muscle retraction. Muscle retraction downregulated AChR, desmin, PSD 95, and GAP 43, whereas IM steroid injection attenuated the adverse effects. The restoration of muscle morphology and decreased fibrosis were also facilitated by IM dexamethasone. IM steroid injection appears to protect against retraction damage in paraspinous muscle following spinal surgery. IM steroid paraspinous muscle injection may provide beneficial effects in spinal operations.

Dengue virus (DENV) affects not only peripheral immune cells but also hematopoietic progenitors in the bone marrow, particularly megakaryocytic precursors, which contribute to the thrombocytopenia characteristic of the disease. In this study, we evaluated the relationship between the differentiation status of the megakaryocytic lineage and its permissiveness and antiviral response to DENV. Our results demonstrate that the erythroid–megakaryocytic precursor (K562 cells) was more permissive to DENV infection than megakaryoblasts, as evidenced by immunofluorescence, flow cytometry, and quantification of viral particles. The antiviral response in K562 cells peaked at three days post-infection, with maximal expression of genes associated with the type I interferon (IFN-I) pathway. In vitro-induced differentiation of K562 cells reduced the initial susceptibility to DENV and enhanced the expression of Toll-like receptor 3 (TLR3) and the type I interferon receptor (IFNAR1), accelerating and intensifying IFN-β secretion, and increasing the expression of OAS2 and IRF3. Furthermore, pretreatment of K562 cells with recombinant IFN-β significantly reduced viral replication from the first day post-infection. Collectively, these findings demonstrate for the first time that the differentiation status of erythroid–megakaryocytic progenitor critically shapes their antiviral response and underscore the central role of IFN-β in the early restriction of DENV infection.