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To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway.Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot.Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1β and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1β and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different (0.05).In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Gladiolin is a novel macrolide antibiotic with promising activity against multi-drug resistant Mycobacterium tuberculosis, isolated from Burkholderia gladioli BCC0238 and it is chemically similar to etnangien, a polyketide discovered from Sorangium cellulosum. In this work, the function of a trans-acting enoyl reductase (ER) from the etnangien polyketide synthase (EtnL) was investigated. It was shown EtnL only interacts with the ACP domain from the module 10 of the etnangien polyketide synthase and functioned identically to GbnE, the corresponding ER from the gladiolin polyketide synthase, which prevents iteration in the module 5 during gladiolin biosynthesis. This indicates it is the differential recognition of the ACP domain in module 5 that is responsible for the iterative or non-iterative nature of the etnangien and gladiolin biosynthesis. \ud \ud The gladiolin polyketide synthase contains a hypothesized non-elongating ketosynthase (KS) domain in its final module. Interestingly, the active site His residue in the HSVGS motif, responsible for initiating chain extension and commonly mutated in previously characterised non-elongating KS domains, is intact. In this work, the non-elongating nature of this KS domain was experimentally verified and the downstream thioesterase (TE) domain was shown to efficiently hydrolyze malonyl thioesters from the interspersed ACP domain. Thus, the TE domain removes malonyl thioesters from the ACP domain before chain elongation can occur. Through a combination of X-ray crystallography, phylogenetic analysis, and site-directed mutagenesis, a Gly residue in the active site was found to play a key role in enabling the TE domain to hydrolyse malonyl thioesters. This residue was conserved in all TE domains appended to trans-AT PKS modules containing a non-elongating KS domain and an associated ACP domain. \ud \ud Gladiostadin is a 2-acyl-4-hydroxy-3-methylbutenolide (AHMB)-containing polyketide recently discovered as the metabolic product of a trans-AT PKS in B. gladioli with promising anticancer activities. The formation of the AHMB moeity involves a phosphate migration reaction catalysed by a unique phosphamutase (GdsG) belonging to the haloacid dehalogenase (HAD) enzyme family. In this study, the X-ray crystal structure of a GdsG homologue (SabP) from the biosynthesis of the AHMB-derived SAB signaling molecules was determined and active site residues proposed to play an important role in catalysis were investigated using site directed mutagenesis.\ud