Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.
The integrity of transplanted mesenchymal stem cells (MSCs) for cardiac regeneration is dependent on cell-cell or cell-matrix adhesion, which is inhibited by reactive oxygen species (ROS) generated in ischemic surroundings after myocardial infarction. Intracellular ROS play a key role in the regulation of cell adhesion, migration, and proliferation. This study was designed to investigate the role of ROS on MSC adhesion. In H 2 O 2 treated MSCs, adhesion and spreading were inhibited and detachment was increased in a dose-dependent manner, and these effects were significantly rescued by co-treatment with the free radical scavenger, N-acetyl-L-cysteine (NAC, 1 mM). A similar pattern was observed on plates coated with different matrices such as fibronectin and cardiogel. Hydrogen peroxide treatment resulted in a marked decrease in the level of focal adhesion-related molecules, such as phospho-FAK and p-Src in MSCs. We also observed a significant decrease in the integrin-related adhesion molecules, aV and b1, in H 2 O 2 treated MSCs. When injected into infarcted hearts, the adhesion of MSCs co-injected with NAC to the border region was significantly improved. Consequently, we observed that fibrosis and infarct size were reduced in MSC and NAC-injected rat hearts compared to in MSC-only injected hearts. These results indicate that ROS inhibit cellular adhesion of engrafted MSCs and provide evidence that the elimination of ROS might be a novel strategy for improving the survival of engrafted MSCs.
Fermentative production of cadaverine from renewable resources may support a sustainable biorefinery process to produce carbon-neutral nylons such as biopolyamide 510 (PA510). Cost-competitive production of cadaverine is a key factor in the successful commercialization of PA510. In this study, an integrated biological and chemical process involving cadaverine biosynthesis, purification, and its polymerization with sebacic acid was developed to produce bio-PA510. To stably express ldcC from Escherichia coli in an engineered Corynebacterium glutamicum PKC strain, an expired industrial L-lysine-producing strain, ldcC, was integrated into the chromosome of the C. glutamicum PKC strain by disrupting lysE and controlling its expression via a strong synthetic H30 promoter. Cadaverine was produced at a concentration of 103.78 g/L, the highest titer to date, from glucose by fed-batch culture of this engineered C. glutamgicum PKC strain. Fermentation-derived cadaverine was purified to polymer-grade biocadaverine with high purity (99%) by solvent extraction with chloroform and two-step distillation. Finally, biobased PA510 with good thermal properties (T m 215 °C and T c 158 °C) was produced by polymerization of purified cadaverine with sebacic acid. The hybrid biorefinery process combining biological and chemical processes demonstrated in this study is a useful platform for producing biobased chemicals and polymers.
Micro-RNAs (miRNAs) are a class of small non-coding RNAs, recently emerged as a post-transcriptional regulator having a key role in various cardiac pathologies. Among them, cardiac fibrosis that occurs as a result from an imbalance of extracellular matrix proteins turnover and is a highly debilitating process that eventually lead to organ dysfunction. An emerging theme on is that miRNAs participate in feedback loop with transcription factors that regulate their transcription. NF-κB, a key transcription factor regulator controls a series of gene program in various cardiac diseases through positive and negative feedback mechanism. But, NF-κB mediated miRNA regulation in cardiac fibrosis remains obscure. Bioinformatics analysis revealed that miR-26a has targets collagen I and CTGF and possesses putative NF-κB binding element in its promoter region. Here, we show that inhibition of NF-κB in cardiac fibroblast restores miR-26a expression, attenuating collagen I, and CTGF gene expression in the presence of Ang II, conferring a feedback regulatory mechanism in cardiac fibrosis. The target genes for miR-26a were confirmed using 3'-UTR luciferase reporter assays for collagen I and CTGF genes. Using NF-κB reporter assays, we determine that miR-26a overexpression inhibits NF-κB activity. Finally, we show that miR-26a expression is restored along with the attenuation of collagen I and CTGF genes in cardiac specific IkBa triple mutant transgenic mice (preventing NF-κB activation) subjected to 4 weeks transverse aortic banding (TAC), compared to wild type (WT) mice. The data indicate a potential role of miR-26a in cardiac fibrosis and, offer novel therapeutic intervention.
BackgroundIn the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs.ResultsHere we describe the metabolic engineering of baker’s yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella entericaSEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L−1 3HP with a yield of 13 % C-mol C-mol−1 glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by 13C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed.ConclusionsIn this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0451-5) contains supplementary material, which is available to authorized users.
BackgroundApocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants—like raspberries and roses—by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid β-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP.ResultsIntegration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of β-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased β-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of β-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase.ConclusionsAn efficient β-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene—the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0273-x) contains supplementary material, which is available to authorized users.
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