Persistent post-surgical pain is a difficult clinical problem. In this study, we intend to explore the mechanism underlying the persistent post-surgical pain in SMIR (skin/muscle incision and retraction) rats. First of all, the expression of membrane protein Nav1.7 and p-p65 (Phosphorylation of p65) were detected in ipsilateral L4–6 DRGs of SMIR rats by western-blot and immunostaining. Then with ProTx-II (Nav1.7 blocker) or PDTC (p65 inhibitor) were intrathecally injected while the change of Nav1.7 expression and mechanical withdrawal threshold were detected. Finally chromatin immunoprecipitation assay method was used to detect whether could p-p65 bind in the Nav1.7 gene promoter region directly. The results shows that mechanical hyperalgesia occurs following SMIR model, from 5 day (d) and lasted more than 20d after surgery. Meanwhile, the expression of Nav1.7 was up-regulated at 10d, 15d and 20d after surgery compared with naïve group. The expression of p-p65 was up-regulated at 10d and 15d compared with incision group. The mechanical hyperalgesia induced by SMIR was reversed after blocking Nav1.7 or inhibiting p65. Furthermore, Nav1.7 expression was down-regulated when p-p65 was inhibited and p-p65 could combine with the Nav1.7 gene promoter region directly. Membrane protein Nav1.7 could participate in the peripheral sensitization of persistent post-surgical pain, which may be regulated by p-p65.

Evolutionary mechanisms of substrate specificities of enzyme families remain poorly understood. Plant SABATH methyltransferases catalyze methylation of the carboxyl group of various low molecular weight metabolites. Investigation of the functional diversification of the SABATH family in plants could shed light on the evolution of substrate specificities in this enzyme family. Previous studies identified 28 SABATH genes from the Populus trichocarpa genome. In this study, we re-annotated the Populus SABATH gene family, and performed molecular evolution, gene expression and biochemical analyses of this large gene family. Twenty-eight Populus SABATH genes were divided into three classes with distinct divergences in their gene structure, expression responses to abiotic stressors and enzymatic properties of encoded proteins. Populus class I SABATH proteins converted IAA to methyl-IAA, class II SABATH proteins converted benzoic acid (BA) and salicylic acid (SA) to methyl-BA and methyl-SA, while class III SABATH proteins converted farnesoic acid (FA) to methyl-FA. For Populus class II SABATH proteins, both forward and reverse mutagenesis studies showed that a single amino acid switch between PtSABATH4 and PtSABATH24 resulted in substrate switch. Our findings provide new insights into the evolution of substrate specificities of enzyme families.

Plant enzymes significantly contribute to the rapidly diversified metabolic repertoire since the colonization of land by plants. Carboxylesterase is just one of the ubiquitous, multifunctional and ancient enzymes that has particularly diversified during plant evolution. This study provided a status on the carboxylesterase landscape within Viridiplantae. A total of 784 carboxylesterases were identified from the genome of 31 plant species representing nine major lineages of sequenced Viridiplantae and divided into five clades based on phylogenetic analysis. Clade I carboxylesterase genes may be of bacterial origin and then expanded and diversified during plant evolution. Clade II was first gained in the ancestor of bryophytes after colonization of land by plants, Clade III and Clade IV in ferns which were considered the most advanced seedless vascular plants, while Clade V was gained in seed plants. To date, the functions of carboxylesterase genes in woody plants remain unclear. In this study, 51 carboxylesterase genes were identified from the genome of Populus trichocarpa and further divided into eight classes. Tandem and segmental duplication events both contributed to the expansion of carboxylesterase genes in Populus. Although carboxylesterase genes were proven to enhance resistance to pathogens in many herbaceous species, relevant researches on forest trees are still needed. In this study, pathogen incubation assays showed that overexpressing of six Class VI carboxylesterases in Populus tomentosa, to a greater or lesser degree, reduced colonization of detached leaves by fungus Cytospora chrysosperma. A significant difference was also found in functional divergence patterns for genes derived from different gene duplication events. Functional differentiation of duplicated carboxylesterase genes in Populus was proved for the first time by in vivo physiological analysis. The identification of the potentially anti-fungal PtoCXE06 gene also laid a theoretical foundation for promoting the genetic improvement of disease-resistance traits in forest trees.