Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase ...Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.展开更多
Objective： The present study was designed to use an in vivo rabbit ear scar model to investigate the efficacy of systemic administration of endostatin in inhibiting scar formation. Methods： Eight male New Zealand wh...Objective： The present study was designed to use an in vivo rabbit ear scar model to investigate the efficacy of systemic administration of endostatin in inhibiting scar formation. Methods： Eight male New Zealand white rabbits were randomly assigned to two groups. Scar model was established by making six full skin defect wounds in each ear. For the intervention group, intraperitoneal injection of endostatin was performed each day after the wound healed （about 15 d post wounding）. For the control group, equal volume of saline was injected. Thickness of scars in each group was measured by sliding caliper and the scar microcirculatory perfusion was assessed by laser Doppler flowmetry on Days 15, 21, 28, and 35 post wounding. Rabbits were euthanatized and their scars were harvested for histological and proteomic analyses on Day 35 post wounding. Results： Macroscopically, scars of the control group were thicker than those of the intervention group. Significant differences between the two groups were observed on Days 21 and 35 （p〈0.05）. Scar thickness, measured by scar elevation index （SEI） at Day 35 post wounding, was significantly reduced in the intervention group （1.09±0.19） compared with the controls （1.36±0.28）. Microvessel density （MVD） observed in the intervention group （1.73±0.94） was significantly lower than that of the control group （5.63±1.78） on Day 35. The distribution of collagen fibers in scars treated with endostatin was relatively regular, while collagen fibers in untreated controls were thicker and showed disordered alignment. Western blot analysis showed that the expressions of type I collagen and Bcl-2 were depressed by injection of endostatin. Conclusions： Our results from the rabbit ear hypertrophic scar model indicate that systemic application of endostatin could inhibit local hypertrophic scar formation, possibly through reducing scar vascularization and angiogenesis. Our results indicated that endostatin may promote the apoptosis of endothelial cells and block their release of platelet-dedved growth factor （PDGF） and fibroblast growth factor （FGF）, thereby controlling collagen production by fibroblasts. Blood vessel-targeted treatment may be a promising strategy for scar therapy.展开更多
基金supported by grants from the Ministry of Science and Technology of China(2013CB734001 and 2015CB150600)the National Natural Science Foundation of China(31370097 and 31571281)
文摘Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a^40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.
基金supported by the National Natural Science Foundation of China (No.81272120)the Health Department of the Zhejiang Province (No.2007B086),China
文摘Objective： The present study was designed to use an in vivo rabbit ear scar model to investigate the efficacy of systemic administration of endostatin in inhibiting scar formation. Methods： Eight male New Zealand white rabbits were randomly assigned to two groups. Scar model was established by making six full skin defect wounds in each ear. For the intervention group, intraperitoneal injection of endostatin was performed each day after the wound healed （about 15 d post wounding）. For the control group, equal volume of saline was injected. Thickness of scars in each group was measured by sliding caliper and the scar microcirculatory perfusion was assessed by laser Doppler flowmetry on Days 15, 21, 28, and 35 post wounding. Rabbits were euthanatized and their scars were harvested for histological and proteomic analyses on Day 35 post wounding. Results： Macroscopically, scars of the control group were thicker than those of the intervention group. Significant differences between the two groups were observed on Days 21 and 35 （p〈0.05）. Scar thickness, measured by scar elevation index （SEI） at Day 35 post wounding, was significantly reduced in the intervention group （1.09±0.19） compared with the controls （1.36±0.28）. Microvessel density （MVD） observed in the intervention group （1.73±0.94） was significantly lower than that of the control group （5.63±1.78） on Day 35. The distribution of collagen fibers in scars treated with endostatin was relatively regular, while collagen fibers in untreated controls were thicker and showed disordered alignment. Western blot analysis showed that the expressions of type I collagen and Bcl-2 were depressed by injection of endostatin. Conclusions： Our results from the rabbit ear hypertrophic scar model indicate that systemic application of endostatin could inhibit local hypertrophic scar formation, possibly through reducing scar vascularization and angiogenesis. Our results indicated that endostatin may promote the apoptosis of endothelial cells and block their release of platelet-dedved growth factor （PDGF） and fibroblast growth factor （FGF）, thereby controlling collagen production by fibroblasts. Blood vessel-targeted treatment may be a promising strategy for scar therapy.
