RNAe is a new method that enhances protein expression at the post-transcriptional level. RNAe utility was further explored to improve endogenous protein expression.Transgenic mice were created by targeting RNAe to growth hormone gene into the C57/BL mouse genome by transposon mediated integration; the mice showed a heavier body weight and longer body length compared with normal mice. RNAe can also be used for gene therapy through the delivery of in vitro transcribed RNA.This study takes a further step towards applying RNAe in pharmaceutical approaches by transposon-based transgenic mice model construction and the use of in vitro transcribed RNA transfection assay.

To produce extracellular chiral 3-hydroxyacyl acids (3HA) by fermentation, a novel pathway was constructed by expressing tesB gene encoding thioesterase II into Pseudomonas putida KTOY01, which was a polyhydroxyalkanoate (PHA) synthesis operon knockout mutant. 3HA mixtures of 0.35 g/l consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (3HDD) were produced in shake-flask study using dodecanoate as a sole carbon source. Additional knockout of fadB and fadA genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KTOY01 led to the weakening of the β-oxidation pathway. The fadBA and PHA synthesis operon knockout mutant P. putida KTOY07 expressing tesB gene produced 2.44 g/l 3HA, significantly more than that of the β-oxidation intact mutant. The 3HA mixture contained 90 mol% 3HDD as a dominant component. A fed-batch fermentation process carried out in a 6-l automatic fermentor produced 7.27 g/l extracellular 3HA containing 96 mol% fraction of 3HDD after 28 h of growth. For the first time, it became possible to produce 3HDD-dominant 3HA monomers.

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield YPHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the YPHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.

This paper reports two soft lithographic methods, micromolding and hot embossing, to produce biodegradable poly (3-hydroxybutyrate-co-3-ftydroxyhexanoate) (PHBHHx) arrays of microstructures for hosting and culturing cells in a local microenvironment by controlled shape. Silicon masters with high-aspect-ratio microfeatures were fabricated using KOH and DRIE anisotropic etching. These silicon masters were used as molds to construct PHBHHx microstructures using micromolding and hot embossing. Using silicon rather than conventional PDMS as molds allowed microstructures with feature size of 20 μm and height of 100 μm to be realized. PHBHHx microstructures with different configurations including circles, rectangles, and octagons were fabricated to investigate the effects of topography on cell culture. Mouse fibroblast cell lines L929 were cultured on PHBHHx microstructures in vitro to investigate the biocompatibility. This study demonstrates the feasibility of microfabrication of PHBHHx structures with micro-scale feature size using soft lithography, and the results show that PHBHHx microstructures can be created to mimic cellular microenvironment for cell culture, providing a convenient means to investigate relationships of microstructures and cell functions.

As a prerequisite for tissue engineering applications, researchers must understand the effect on local cell types of the degradation products of biodegradable polymers. Polyhydroxybutyrate (PHB) has received special interest as an implant material, because it degrades to release a normal component of blood and tissue, D,L-β-hydroxybutyrate (HB). We report that HB (0.02 g/l) promoted cell proliferation in cultured L929 cells plated at high cell density (1×105 cells/well) but not lower cell densities. While HB did not affect cell cycle progression, it significantly inhibited cell death. HB treatment prevented necrosis, reducing cell membrane permeability 4 h following serum withdrawal from the medium, and for all subsequent time points. In summary, HB promotes proliferation of L929 cells in high-density cultures by preventing apoptotic and necrotic cell death. This property makes biodegradable polymers containing HB, such as PHBHHx, attractive candidates for tissue engineering applications, especially those requiring the regeneration of large numbers of cells.

In this paper, comprehensive characteristics including cell attachment, cell proliferation status, cell cycle progression and phenotypic changes of smooth muscle cells from rabbit aorta (RaSMCs) were studied on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 0–20% HHx (mol%) in comparison with tissue culture plates (TCPs). Results demonstrated that RaSMCs adhered better on PHBHHx containing 12% HHx (12%HHx) although they proliferated better on 20%HHx-containing PHBHHx films (20%HHx). This was explained by the difference in cell cycle progression observed using flow cytometry, as it was found that only 20%HHx-containing polymer could maintain the normal cell cycle evolution as TCPs did after 3 d incubation. The highest expression level and typical spindle-like distribution of α-actin on 20%HHx-containing polymer were characterized as the contractile-like phenotype, suggesting that RaSMCs tended to differentiate rather than proliferate compared to the cells grown on 12%HHx polymer. Results obtained above suggested that 20%HHx was suitable for RaSMCs proliferation, leading to its change to contractile phenotype. This study extends the potential applications of PHBHHx in SMCs-related graft scaffold fabrication for tissue engineering.

Films made of poly (3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx) consisting of 5%, 12% and 20% hydroxyhexanoate (HHx), respectively, were evaluated for biomedical application in comparison with poly (l-Lactide) (PLA). With the increase of HHx content in PHBHHx, the polymer surface properties changed accordingly. P(HB-co-20%-HHx) had the smoothest surface while PHB surface was most hydrophilic among the evaluated PHB and all the PHBHHx. All PHBHHx also showed strong protein affinity and biocompatibility. It was found that fibroblast and osteoblast had different responses to these polymers: fibroblast cells favored P(HB-co-20%-HHx), yet osteoblast cells preferred P(HB-co-12%-HHx). PHB and all PHBHHx appeared to have better biocompatibility for fibroblast and osteoblast compared with PLA. Polymers possessing diferent surface properties may help meet different cellular requirements. Combined with their good mechanical properties for elongation and adjustable biocompatibility, PHBHHx may meet the needs of growth requirements of different tissues and cells.

Random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate, short as PHBHHx, was surface modified by ammonia plasma treatment and/or fibronectin coating, respectively. The improved results were demonstrated by better growth of human umbilical vein endothelial cells (HUVECs) and rabbit aorta smooth muscle cells (SMCs) on the surface of ammonia plasma-treated PHBHHx coated with fibronectin (PFn-PHBHHx), compared with the fibronectin-coated (Fn-PHBHHx) or uncoated PHBHHx, respectively, although XPS analysis and ELISA demonstrated higher fibronectin adsorption on Fn-PHBHHx than on PFn-PHBHHx. Confocal microscopy observation showed that the specific co-localization of fibronectin with F-actin was impaired on PFn-PHBHHx, while it was almost lost on Fn-PHBHHx compared with pristine PHBHHx or plasma-treated PHBHHx (P-PHBHHx). These were attributed to the generation of new nitrogen- and oxygen-containing groups on the PHBHHx surface by the ammonia plasma treatment, which led to increased polar components that enhanced polymer surface energy and hydrophilic properties on P-PHBHHx. The most prominent effect of PFn-PHBHHx was its stimulation of HUVECs proliferation. HUVECs on PFn-PHBHHx formed a confluent monolayer after 3 days of incubation, while SMCs were unable to form a sub-confluent layer. The above evidences revealed that PFn-PHBHHx would benefit endotheliazation rather than SMCs proliferation. We therefore believed that PFn-PHBHHx would be a promising material as a luminal surface of vascular grafts.

Targeted therapy for cancer is a research area of great interest, and magnetic nanoparticles (MNPs) show great potential as targeted carriers for therapeutics. One important class of cancer biomarkers is microRNAs (miRNAs), which play a significant role in tumor initiation and progression. In this study, a cascade recognition system containing multiple plasmids, including a Tet activator, a lacI repressor gene driven by the TetOn promoter, and a reporter gene repressed by the lacI repressor and influenced by multiple endogenous miRNAs, was used to recognize cells that display miRNA signals that are characteristic of cancer. For this purpose, three types of signal miRNAs with high proliferation and metastasis abilities were chosen (miR-21, miR-145, and miR-9). The response of this system to the human breast cancer MCF-7 cell line was 3.2-fold higher than that to the human breast epithelial HBL100 cell line and almost 7.5-fold higher than that to human embryonic kidney HEK293T cells. In combination with polyethyleneimine-modified MNPs, this recognition system targeted the tumor location in situ in an animal model, and an ≃42% repression of tumor growth was achieved. Our study provides a new combination of magnetic nanocarrier and gene therapy based on miRNAs that are active in vivo, which has potential for use in future cancer therapies.