Abstract:Synthetic biology is an emerging biological discipline in the 21st century. It focuses on the combination of biological sciences and engineering sciences. The engineering formalism was used to design, modify and assemble cellular components into biological systems in the "bottom-up" way that comes up from "unit" to "parts" then to "System". Synthetic biology is a multidisciplinary study of molecular and cellular biology, evolutionary systematics, biochemistry, informatics, mathematics, computer and engineering. Recently, synthetic biology has been redefined as for: (I) the design and construction of new biological parts, devices, and systems that do not already exist in the nature, and (II) the re-design of existing, natural biological systems for novel function or products. As an established post-genome method, synthetic biology emphasizes on the synergistic integration of computational biology and experimental biology for the existing or new life forms. It must be noticed that the elements used in synthetic biology for new cellular structure or function may not only be genes, nucleic acids and other biological components, but also be chemical, mechanical and physical components. This article is a review for the most recently progress in the fundamental research and applications in synthetic biology. In particular, attentions were paid on synthetic biology study on the programming at the DNA level, molecular modification, metabolic pathways, regulatory networks, and industrial biotechnology and other aspects.

Abstract:The objectives of synthetic biology include to understand life through synthesis and to engineer complex biological systems based on general principles of modern engineering sciences. The engineering goal relies on the availability of molecular parts with diverse functions that can be predictably integrated, regulated and reused. These parts may include proteins, RNA, DNA and their complexes. Based on the understanding of molecular mechanisms and judicious combinations of rational design and laboratory evolution, specific interactions, regulatory functions and activities of biomolecules can be artificially changed or created. This comprises an important strategy to artificially regulate and reprogram existing biological systems. It also lays the foundation for the bottom-up design and construction of artificial biological systems of increasing complexity.

Abstract:Membrane proteins play crucial roles in substance transportation, energy transformation and signal transduction. Three dimensional structural determination of membrane protein is significantly important for its biological function study and rational drug design. The molecular structures of membrane proteins are mainly determined via X-ray single crystal diffraction technique, for which high quality membrane protein crystals are required. However, due to its amphipathicity, the membrane proteins are hard to crystallize, resulting in difficulties in the structural determination. In recent years, some methods (such as in surfo methods and in cubo methods) have been developed specifically for crystallizing membrane proteins. The progresses on crystallization methodology of membrane proteins are reviewed, and the prospective in this field are also discussed.

Abstract:Affibodies are a new class of engineered affinity proteins derived from staphyloccocal protein A (SPA) B domain. B domain is composed of 58 amino acids with a molecular mass of 6.5 ku and has a three-helical bundle structure. Thirteen amino acid positions in helix 1 and helix 2 of B domain can be randomly mutated to construct an affibody library without obvious compromise in its structural stability. Theoretically, this library contains affibodies specific to any given target. Using affinity panning, a specific affibody can be obtained. Affibody has some functional similarities with antibody. However, compared with antibody, affibody has some outstanding properties. Affibody can be obtained by in vitro selection and can be produced in large scale by chemical synthesis or prokaryotic expression. The smaller molecular size confers its higher tissue penetrativity and faster circulation clearance. Affibody has a higher physical and chemical stability than antibody. In addition, affibody can be cross-liked with or fusion co-expressed with reporter molecules such as fluorescent protein, biotin and so on. This class of engineered affinity proteins has been used in detection, separation, and purification of target proteins and, might be extensively applied in experimental diagnosis, molecular imaging and targeted therapeutics in the future.

Abstract:Hepatitis C virus (HCV) F protein has been identified for more than ten years, but its functions remain unclear. In order to understand its roles in viral replication and infection, in this study 5 stop codons (nt406T-A, nt433T-A, nt472G-A, nt479/481G-A, G-A, nt613C-A) were introduced in plasmid J6JFH1 to construct J6JFH1/ΔF which interrupt F protein expression, and then monitor the viral RNA replication and viral protein expression. Our data showed that the five mutations did not affect core gene replication and expression. The virus strain J6JFH1/ΔF significantly reduced in the expression of viral proteins in transfected cells compared with wild-type J6JFH1, and viral RNA levels dropped by nearly 95% (J6JFH1/ΔF (7.39×10⁶±2.54×10⁴) vs wild type (1.17×10⁸±1.46×10⁷), P < 0.001). The infectivity of virus released into the supernatant significantly reduced similarly. Further study found that these five mutations changed secondary structure of HCV core gene. In order to further explore the underlying reasons for the weakening of J6JFH1/ΔF virus replication and infection, we constructed five separate mutant viruses based on J6JFH1, respectively, and form J6JFH1m1 nt406T-A, J6JFH1m2 nt433T-A, J6JFH1m3 nt472G-A, J6JFH1m4 nt479/481G-A, G-A, J6JFH1m5 nt613C-A. These 5 virus mutants do not influence the secondary structure of the core region, while J6JFH1m1, J6JFH1m2, J6JFH1m3, J6JFH1m4, J6JFH1m5 respectively stopped F protein expression shifting from nt374-383, nt417-419, nt436-445, nt474-477, nt597-605, in which J6JFH1m5 stopped all reported forms of F protein. By in vitro transcription and transfection into Huh7.5.1 cells, 48 hours post-transfection, the detection of intracellular viral protein NS5A expression levels, viral RNA in transfected cells, and virus titers in supernatant were carried out. The results showed that no significant differences were found among groups five separate mutants and wild type. These results indicated that the lack of F protein itself did not affect HCV viral replication and virion assembly and infectivity. All the results were seemed to result from the secondary structure of the core gene, but the mechanism was yet to be further studied. We concluded that HCV F protein deficiency did not affect translation of viral replication and packaging and release of virus particles, the core gene secondary structure changes had a great impact on viral replication and protein expression.

Abstract:The aim of this present work was to investigate whether curcumin reverses the adriamycin-resistance of thermotolerant hepatocarcinoma cell line and the underlying mechanisms. Cytotoxicity was evaluated by MTT assay. Apoptosis was determined by flow cytometer using propidium iodide staining. The accumulation of intracellular adriamycin was measured by high-performance liquid chromatography. The expressions of P-glycoprotein (P-gp), heat shock protein 70 (Hsp70), and caspase-3 were analyzed by Western blotting. The thermotolerant hepatocarcinoma cell line HepG2/TT was resistant to adriamycin-induced cytotoxicity and apoptosis. Curcumin, at 5 μmol/L, 10 μmol/L, and 20 μmol/L, decreased the IC₅₀ of adriamycin to thermotolerant HepG2/TT cells and enhanced adriamycin-induced apoptosis in HepG2/TT cells in a concentration-dependent manner. The levels of P-gp and Hsp70 in HepG2/TT cells were obviously higher than that in HepG2 cells. Treatment with curcumin (10 μmol/L) for 24 h significantly reduced the levels of P-gp and Hsp70 in HepG2/TT cells. The accumulation of intracellular adriamycin in HepG2/TT cells was markedly lower than that in HepG2 cells and curcumin (10 μmol/L for 3 h) significantly increased the level of intracellular adriamycin accumulation in HepG2/TT cells. HepG2/TT cells obviously inhibited caspase-3 activation triggered by adriamycin, but co-treatment with 10 μmol/L of curcumin for 24 h significantly augmented the activation of caspase-3 in HepG2/TT cells treated with adriamycin. Curcumin overcomes the adriamycin-resistance of thermotolerant HepG2/TT cells by promoting adriamycin-triggered caspase-3 activation through down-regulating the activity and expression of P-gp and the expression of Hsp70.

Abstract:Embelin, a small molecule XIAP (X-linked inhibitor of apoptosis protein) inhibitor, can inhibit the expression and activation of XIAP, thereby removing the antiapoptotic ability of XIAP and resulting in apoptosis process smoothly. This study is attempt to explore the mechanism of the inhibition to hepatocarcinoma cell Bel-7404 growth by Embelin. After treated with different-concentration of Embelin, the morphological change, formation of apoptotic bodies, cells viability, cell apoptosis and the apoptosis-related protein expression in Bel-7404 cells were assessed by fluorescence microscope, Hochest33342 staining, MTT assay, Annexin Ⅴ/PI flow cytometry and Western blotting et al. The results indicated that Embelin remarkably inhibited human hepatocarcinoma cell Bel-7404 growth. Further experiments found that the mechanism of tumor cell growth inhibition by Embelin was due to the cell apoptosis with the activation of caspase apoptotic pathway and blockage of NF-κB signal pathway. The studies laid a base on the further treatment of human hepatoma carcinoma by using Embelin.

Abstract:To study the effect of CXCR4 antagonist AMD3100 on the atherosclerotic lesion, the expression of TNF-α and NF-κB and SDF-1α/CXCR4, so as to approach the specific role and possible mechanisms of SDF-1α/CXCR4 on atherosclerosis. 36 male apoE^-/- mice, 8 weeks old, randomly divided into three groups: ①normal food group, ② high fat group, ③AMD3100 group. Animals from high fat group and AMD3100 group were fed with western food which including 15% fat and 0.25% cholesterol. After feeding 12 weeks, all mice were anesthetized by 0.2～0.3 ml Urethane (20%) and removed eyeball in order to obtain blood preparation. Serum lever of SDF-1α was measured by ELISA. Serum triglyceride (TG), total cholesterol (TC), and high density lipoprotein cholesterol (HDL-C) were determined by commercially enzymatic methods. The Hematoxylin/Eosin dyeing of paraffin section was used to detect the area of atherosclerotic plaque of apoE^-/- mice aortic root transaction. The expression of CXCR4, TNF-α and NF-κB was analyzed by real time RT-PCR and Western blot, respectively. As a result, AMD3100 treatment resulted in a significant increase of atherosclerotic lesion area and the expression of TNF-α and NF-κB in apoE^-/- mice. Serum TG, TC, HDL-C and LDL-C concentrations were not markedly changed. AMD3100 reduced SDF-1α serum lever and CXCR4 expression on artery wall. It can be concluded that, atherosclerotic lesions in apoE-/- mice were aggravated by long term administration of CXCR4 antagonist AMD3100. Possible mechanisms of this action for AMD3100 are associated with the up-regulation pro-inflammatory factors TNF-α and NF-κB and down-regulation SDF-1α/CXCR4 axis.

Abstract:Essential polyunsaturated fatty acids can not be synthesized in mammals due to the lack of Δ-12 and ω-3 fatty acid desaturases. Expressing ω-3 fatty acid desaturases in transgenic mammals could convert long chain n-6 polyunsaturated fatty acids to n-3 polyunsaturated fatty acids, significantly reducing the level of long chain n-6 polyunsaturated fatty acids. In this study, the muscle-specific transgenic mice expressed Caenorhabditis elegans FAT-1 and FAT-2 genes, which encode ω-3 and Δ-12 fatty acid desaturases respectively and human catalase hCAT gene, based on "self-cleaving" 2A peptides, and they were generated by microinjection. Fatty acids in skeletal muscle were analyzed by gas chromatography. The level of total n-3 polyunsaturated fatty acids in transgenic mice was 2.6-fold higher than that in wild type mice, while there was no significantly difference in the level of total n-6 polyunsaturated fatty acids between transgenic and wild type mice. But the skeletal muscle tissue of transgenic mice had a dramatically reduced ratio of n-6/n-3 fatty acids (P < 0.01). In addition, the expression level of human catalase was identified by Western blot, and the catalase activity of hCAT in skeletal muscle of transgenic mice was significantly higher than that of wild type mice (P < 0.01).

Abstract:Co-delivery of B-domain-deleted FⅧ(BDD-FⅧ) heavy and light chain genes by dual-vector system is one of strategies to overcome packaging constraint of adeno-associated virus vectors (AAV), but leading to chain imbalance because of inefficient secretion of heavy chain. We hypothesize that a disulfide linking between the heavy and light chains can improve the efficacy of dual-vector co-delivery of BDD-FⅧ gene. Here, we plan to achieve this by making Cys mutations at the Met662 in A2-domain of heavy chain and Asp1828 in A3-domain of light chain. By co-transfecting cultured COS-7 cells transiently with mutant heavy and light chain genes, a disulfide cross-linked heavy and light chain was observed by Western blotting. The secretion of heavy chain and coagulation activity were up to (109±13) μg/L and (0.65±0.12) U/ml respectively analyzed by ELISA and Coatest method greater than that of two chains of wild-type BDD-FⅧ gene co-transfected cells((71±11) μg/L and (0.35±0.05) U/ml. It suggests that inter-chain disulfide linking could efficiently improve efficacy of dual-vector delivery of the BDD-FⅧ gene. It provided evidence for ongoing in vivo BDD-FⅧ gene delivery by dual-AAV vector.

Abstract:Skin is the largest organ of the body and is a potential route of exposure to sunscreens and cosmetics containing nanoparticles, but the permeability of the skin to these nanoparticles is unknown. In this paper, we studied the transdermal delivery capacity through mouse skin of water-soluble CdSeS quantum dots (QDs) and the deposition of these QDs in the body. QD solution was coated on the dorsal hairless skin of male ICR mice. Fluorescence microscope was used to observe the deposition of QDs in mouse skin and heart, liver, spleen, lung and kidney. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to measure the ¹¹¹Cd concentration to indicate the concentration of QDs in plasma and organs. The fluorescence images show that QDs can penetrate into the dermal layer and deposit in the organs through blood circulation. ICP-MS result indicates that QDs deposit seriously in liver and kidney, and they are difficult to clear. ¹¹¹Cd concentration is still more than 14 ng/g in kidney after 5 days. These results suggest that QDs has in vivo transdermal delivery capacity through mouse skin and is harmful to the liver and kidney.

Abstract:The extractive electrospray ionization (EESI) technique is an emerging soft ionization technique and it can be used for the sensitive detection of large molecules and small molecules in solid, liquid, gas and viscous samples without sample pretreatment. Based on the brief introduction of the mechanism of EESI, its applications on the protein analysis were reviewed in detail. Compared to the commercial electrospray ionization (ESI) source, EESI maintains the native structure of protein and thus the biological activity of the protein to the most extent, which shows it can be potentially applied in the fields of preparation of protein microarray, high resolution hydrogen/deuterium exchange mass spectrometry (structure analysis of protein), and metrology for protein.