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The goal of this study was to investigate the tissue performance of bladder following stretched electrospun silk fibroin matrix (SESFM) implantation compared with bladder acellular matrix (BAM). We compared SESFM with BAM based on porosity and pore size. Scaffolds were separately transplanted into opposite walls of the bladder of 30 rabbits after stripping the bladder mucosa and smooth muscle (1.5 × 2.0 cm(2)). Gross anatomical observation, histological analysis and muscle contractility studies were performed at 2, 4, and 8 weeks post-op. SESFM has higher porosity and larger pore size compared with BAM (p < 0.05). At 2 weeks, the presence of vesical calculus was evident in 7/10 rabbits. Histological analysis showed that SESFM and BAM promoted similar degree of urothelium regeneration (p > 0.05). However, SESFM promoted a higher degree of smooth muscle and vessel regeneration compared to BAM (p < 0.05). In addition, muscle strips supported by SESFM displayed higher contractile responses to carbachol, KCl, and phenylephrine compared with BAM. At 8 weeks, both matrices elicited similar mild acute and chronic inflammatory reactions. Our results demonstrated that SESFM has greater ability to promote bladder tissue regeneration with structural and functional properties compared to BAM, and with similar biocompatibility.

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As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the K m values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.Key words 4-coumarate : coenzyme A ligase (4CL); isoflavonoid; Pueraria lobata; puerarin Human have used Pueraria lobata as a herbal drug for many years.1,2) Its pharmacological effects are due to the presence of isoflavonoids, which include puerarin, daidzin, and other related metabolites. Puerarin exhibits diverse medicinal properties including hypotensive, 3) hypolipidemic, 4) hypoglycemic, 5) anti-oxidant, 6) anti-ischaemia, 7) vasodilation 8) and estrogen-like effects.9) As an isoflavonoid, the biosynthesis of puerarin should derive from phenylpropanoid metabolism, in which the enzymes in the early steps such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate : coenzyme A ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), and chalcone isomerase (CHI) are conserved in many plant species. Although puerarin was proposed to exhibit diverse pharmacological activities, the genes for the enzymes involved in the pathway have limitedly been isolated and characterized from P. lobata. As part of understanding the isoflavanoid metabolism in P. lobata at the molecular level, we have undertaken standard amplifications of cDNAs on PAL, C4H, 4CL, CHS, CHR, CHI and isoflavone synthase (IFS) by degenerate primers from the P. lobata roots that are deemed as the source tissues mostly accumulating puerarin. Interestingly, the partial cDNAs corresponding to two divergent 4CLs, designated as Pl4CL1 and Pl4CL2, were amplified, and showed only 65% amino acid sequence identity between each other. In plants, 4CLs usually have many isoenzymes, e.g. structurally and functionally different 4CLs were discovered in Arabidopsis thaliana, 10) Glycine max, 11) Lithospermum erythrorhizon 12) , Lolium perenne, 13) Oryza sativa, 14)Phyllostachys edulis, 15) Populus tremuloides, 16) Panicum virgatum, 17)...