Polydimethylsiloxane (PDMS) micropillar arrayed topographic substrates were fabricated, with dimensions of 4 and 10μm in nominal pillar diameter, 4 and 7μm in spacing and 4μm in height, to investigate the expression and responsiveness of transient receptor potential (TRP) V1 and TRPV4 channels of HepG2 cells cultured on the substrates. Quantitative real-time PCR (qRT-PCR) analysis revealed that the mRNA levels of TRPV1 and TRPV4 were significantly up-regulated in the cells grown on all four topographic substrates, when compared with the cells grown on the flat PDMS substrates. The presence of TRPV1 and TRPV4 proteins in HepG2 cells was confirmed with Western blotting, and similar up-regulation of these two channel proteins by the substrate topography also revealed by stronger immunofluorescence staining. Subsequently, the channel responsiveness of TRPV1 and TRPV4 was quantified by using the calcium inflow-responding magnitudes and percentages of cells having a defined responding magnitude upon stimulation by the channel agonists capsaicin and 4-α-phorbol-12, 13-didecanoate(4αPDD), respectively. Herein, Calcium Green-1 as a fluorescent indicator was employed in its dynamic assessment by confocal laser scanning microscopy. The results displayed that upon stimulation by the channel agonist, the calcium influx through TRPV1 exhibits a dynamic characteristic of rapid desensitization with a transient within 25 seconds, and either the relative fluorescence responding amplitudes or the percentages of responsive cells on the topographic substrates are greater than those observed from the cells on the flat substrates. By contrast, stimulation by the TRPV4 agonist caused a calcium response with much slower recovery within 5 minutes, and both the relative fluorescence responding amplitudes and the percentages of responsive cells on the topographic substrates are higher than those of the cells on the flat substrates. Collectively, these data indicate that the TRPV-mediated ion signaling elicits an important role in the cellular phenotypic and functional regulation by the substrate topography.
Cervical cancer is the second most common malignant tumor in women around the world. The expression of miR-124 is significantly down-regulated in cervical cancer. The results showed that exogenous expression of miR-124 could remarkably suppress the proliferation and migration of cervical cancer cells. Luciferase analysis showed that CUB domain containing protein 1 gene (Cdcp1) is a direct down-stream target of miR-124 and restoration of CDCP1 counteracted the tumor suppressive effects of miR-124. The results have a great potential for not only the understanding of the relationship between miRNA and cervical cancer, but also microRNA-based anti-cervical cancer therapy.
Objective: To analyze the effect of SATB2 on BMP9-induced osteogenic differentiation of C2C12 mesenchymal stem cells. Methods: C2C12 cells were treated with ad-BMP9, and then the expressions of SATB2 at the gene and protein level were detected with RT-PCR and Western blot, respectively. Afterwards C2C12 cells were treated with ad-SATB2 or/and BMP9, then ALP activity was detected by quantitative and staining assay, and calcium deposition was detected by Alizarin Red S staining. Results: BMP9 can enhance the expression of SATB2 at the gene level and at the protein level in C2C12 cells. Both ALP activity and calcium deposition of C2C12 cells treated with adenovirus SATB2 or/and BMP9 expressed higher than those of controls. Conclusions: SATB2 can promote BMP9-induced osteogenic differentiation of C2C12 mesenchymal stem cells
Objective: To construct the eukaryotic vector for E6 gene of papilloma virus (HPV16) specifically expressed in skin and to identify the mRNA or protein of E6 in transgenic mice. Methods: pINV promoter and HPV16-E6 were amplified by polymerase chain reaction (PCR), and the obtained segments were sequentially digested and inserted to pcDNA3.1(－) vector removed CMV promoter, acquiring dpcDNA3.1(－)-pINV-E6 vector. The transgenic mice were prepared by micro-injection, while the RT-PCR, Western blot and immunochemistry were used to detect the expression of E6 in transgenic mice. Results: The dpcDNA3.1(－)-pINV-E6 vector was correctly connected by sequencing. Then two positive mice carrying exogenous gene were produced among 31 experimental mice. The F1 generation was got by transgenic mice mating with the wild type mice. The E6 mRNA or protein was specifically positive in skin of the transgenic mice by RT-PCR, Western blot or immunochemistry. Conclusion: The pINV-E6 transgenic model mice were successfully built with E6 gene specifically expressed in mouse skin. This would lay a solid foundation for the further study of the role of E6 in cancer.
Objective: To further compare the ferrichrome binding characteristics between wild type FtsB and mutant Y137A, W204A in Streptococcus pyogenes, and to further determine the ferrichrome binding sites. Methods: Wild type FtsB and Y137A, W204A mutant proteins were prepared. Their ferrichrome binding capacities were compared by ICP-MS and ITC; Their ferrichrome reduction rates were measured by UV-vis spectrometer using Na2S2O4 as the reductive agent; Their ferrichrome binding stabilities were analyzed via thermal and GuHCl denaturation. Results: The binding affinity and stability of the mutants Y137A and W204A with ferrichrome were both lower than that of wild-type FtsB. Whereas their ferrichrome reduction rates were higher than wild-type FtsB. Thus the residues Try137 and Trp204 play an important role in ferrichrome binding of FtsB. Conclutions: A valuable information for the understanding of ferrichrome transport in bacteria was provided, which may be helpful for the development of novel strategies in the control of bacterial infection.
Objective:To investigate the effects of different carbon sources on the high density fermentation of Pichia pastoris and the expression of HPV16_L1 protein under the control of TEF-1 promoter. Methods:Pichia pastoris cells were cultured in 1.5L fermentors using media supplied respectively with a given carbon source. The dynamic curve of cell growth was obtained by sample collection and wet weight detection at designated time points, and Western blot analysis was employed to detect the expression level of HPV16_L1 protein. Results:Utilizing glycerol as a carbon source, the cells grew to a very high density with a cell wet weight of 510 g/L (OD600 was 189.4), and the expression of HPV L1 kept at a high level within 72 hours of fermentation. Utilizing methanol and dextran as carbon source, the wet weight of fermantation cells reached both to about 220g/L (OD600 values were 75.3 and 77.3 respectively). And in methanol utilization, the expression of L1 protein maintained at a high level during the 144 hours of fermentation; in contrast, in dextran utilization, L1 expression reduced significantly after 60 hours fermentation. Using sorbitol as solo carbon, the cells were not clearly proliferated in bioreactor. The highest yield was obtained by using glycerol as carbon source and the culture was collected after about 72 hours fermentation. Conclusion:The results provided valuable information for achieving high density fermentation of Pichia pastoris and efficient preparation of heterogeneous proteins expressed under the control of TEF-1 promoter, and also laid a foundation for developing HPV vaccine in a more convenient and economic way.
Objective: In order to investigate the enzymatic characteristics of ColR75E collagenase of Bacillus cereus R75E, the colR75E collagenase gene was expressed in E. coli prokaryotic expression system and acquired the recombinant ColR75E collagenase protein with high purity. Methods: The colR75E collagenase gene fragments were amplified using the Bacillus cereus R75E genomic DNA as template. The fragments were cloned into the pET28a vector, constructing recombinant pET28a/colR75E plasmid. Then, the pET28a/colR75E plasmid was introduced into the E.coli BL21(DE3). After IPTG induction, the recombinant ColR75E collagenase protein was purified with Ni-NTA affinity chromatography column. Subsequently, the characteristics of ColR75E collagenase of Bacillus cereus R75E were analyzed by collagen-zymography, collagen hydrolysis activity assay, Km and Vmax calculation and collagen degradative products SDS-PAGE detection. Results: The purified recombinant ColR75E collagenase of Bacillus cereus R75E exhibited the collagen hydrolysis activity, both showing with the obvious negative staining in collagen-zymography gel and progressive degradation of native type I collagen extracted from scales of grass carp with the prolong of treatment time by SDS-PAGE. The specific activity of recombinant ColR75E collagenase was 3.62 U/mg, its Km and Vmax were 25.55 μmol/L (2.93 mg/ml)and 5.71μmol/(mg·min), respectively. The optimum temperature to ColR75E collagenase was 45℃ and the optimum pH was 8.0 .In addition, the activity of recombinant ColR75E collagenase was stable both under the temperature no higher than 50℃ and the pH of buffer from 6.0 to 8.0. The recombinant ColR75E collagenase was activated by adding of Ca2+, but inhibited by adding of Zn2+, Pb2+, Fe2+, Mn2+, respectively. The inhibitory capability of these metal ions was Pb2+> Zn2+> Fe2+> Mn2+, subsequently. The high sensitivity of ColR75E collagenase to EDTA and EGTA further confirmed the ColR75E collagenase was a metallo-proteinase. Conclusion: It is feasible to acquire the recombinant ColR75E collagenase with both high purity and perfect hydrolysis activity in E. coli prokaryotic expression system, which supplies a foundation for the extensive application of ColR75E collagenase in field of medical and food industry.
PCR-based strategy provides economical and efficient application in site-directed mutagenesis (SDM) and DNA assembling such as PCR based vector construction. In these processes, primers are extremely important as they determing amplification efficiency of the target DNA fragments,also influencing circularization of the new plasmids. So this web-based software Primer Spanner (PS) was developed to design primer pairs with partially complementary 5'-ends, which is a special tool for chimeric primer designing. Primers designed by this tool are applied to substitution, insertion and deletion for single-site or multi-site mutagenesis. The performance of the software had been verified by designed experiment and DNA sequencing. The PS primer designing engine is freely accessible at: http://PS1.biocloud.org.cn.
Seamless ligation cloning extract (SLiCE) is a seamless ligation method which utilizes cell extracts to assemble DNA fragments in vitro through recombination reaction. Here, this method was further optimized by identifying the sources of cell extract, enhancing the expression of λ recombination system and adjusting the ratio of fragments used in assembly system. The cell extract from Saccharomyces cerevisiae and Bacillus subtilis were proved to be not suitable for assembly in vitro. The plasmid with λ recombination system was introduced into Escherichia coli DH10B, and the cell extract from this strain was proved to be effective for assembly in vitro. A color-based screen method was constructed to identify positive clones and measure the accuracy in the 3-fragments assembly assay. Finally, a melanin expression cassette, which can be expressed in S. cerevisiae, was assembled via the SLiCE method optimized.
Japanese encephalitis(JE),caused by Japanese encephalitis virus(JEV)infection,is an important viral encephalitis which is prevalent in Asian and Pacific areas.JE endemic areas expanded in recent years.At present there is no specially effective antiviral treatment for JE.Therefore,vaccination and vector control constitute major measures.Sevaral new promising Japanese encephalitis vaccine candidates have been developed. A review on JEV genomic structure and their function, recent advances in the area of Japanese encephalitis vaccine development was given.
Glutathione (GSH) is a major antioxidant, as well as redox and cell signaling regulator. GSH Protects cells from oxidative injury by reducing H2O2, scavenging reactive oxygen and nitrogen radicals. In addition, GSH-induced redox shift with or without ROS subjects some cellular proteins to varied forms of oxidation, further altering the function of signal transduction and transcription factor molecules. A lot of experiments showed that ROS and GSH play important roles in modulating multiple signaling pathways. Fas and TNF-a signaling, NF-κB and mitochondrial apoptotic pathways are focused on. Notably, the depletion of mitochondrial GSH induces increased mitochondrial ROS exposure which impairs bioenergetics and promotes mitochondrial permeability transition pore opening which is critical for cell death. Depending on the extent of mitochondrial damage, NF-κB inhibited and hepatocytes may either undergo different modes of cell death (apoptosis or necrosis) or be sensitized to cell death stimuli (i.e.TNF-α). These processes have been implicated in the pathogenesis of many liver diseases.
Courtship behavior regulated by multiple gene in Drosophila, such as fruitless (fru), dissatisfaction (dsf) and retained (retn), etc. Using of these products that were produce specific products by different splicing pattern to control the courtship behavior of male and female in Drosophila, whose splicing pattern is necessary for courtship behavior and sex determination in both male and female. The molecular regulation of these genes that regulate courtship behavior in Drosophila was reviewed and summarized for providing theoretical basis to further study the courtship behavior and sex determination.
Pichia pastoris expression system is a kind of high efficiency expression system of exogenous protein developed in recent years, and it is promising to express exogenous gene in Pichia pastoris. Although the relatively complete mechanism of gene expression and regulation as well as the capability of performing eukaryotic post-translational modifications of Pichia pastoris expression system has been well known, some case with no expression or the low yield were still existed due to numerous factors of gene and expression system. The aim of the expression system of Pichia pastoris, the optimization of expression vectors, engineering strain and fermentation conditions were reviewed to lay the theory foundation that exogenous gene will be expressed efficiently in Pichia pastoris.
Acetoin (3-hydroxy-2-butanone), widely used mainly in foods as an additive to enhance the flavor and in chemical synthesis as a platform chemical, has broad industrial application prospects. It is classified as one of the 30 platform chemicals that were given the priority to their development and utilization by the U.S. Department of Energy. Compared to traditional chemical synthetic methods, economical, environment-friendly and efficient microbial production of acetoin has significant impetus to the low-carbon economy development of China. Currently, many achievements have been made in the biosynthesis of acetoin. This state-of-the-art review summarized the focus of the researchers' attentions nowadays, briefly described the strains used for acetoin production (especially Class 1 microorganisms), reviewed the latest strategies combining metabolic engineering, evolutionary engineering, cofactor engineering and fermentation engineering for acetoin production, and introduced the technology improvement for recovering processing. These achievements in the last five years were classified and discussed with state-of-the-art views. At last, guidelines for future studies were also proposed. It is pointed out that future research should focus on improvement of strains for acetoin tolerance and developing metabolic engineered class I strains for chiral acetoin production with high yield and productivity. More attention should be paid for the improvement of separation and purification process to lower the cost of downstream processing.
The nitrile converting system can overcome the shortcomings of serious pollutions and unsafe factors of traditional ones, providing new routes for the green chemical engineering and environmental friendly processes. The microbial diversity and biotransformation characteristics of nitrile converting enzymes are presented. NHase gene expression and its tolerance are also discussed.In the near future, an increasing number of novel nitrile converting enzymes will be screened and their potential in the production of useful fine chemicals will be further exploited.
Extensive uses of antibiotics have resulted in rapid growing resistance and emergence of resistant bacterial pathogens in medical availability and clinical practice. Resistance to a variety of antibiotics in microbial pathogens is currently considered as one of the most challenging problems in public health care. However, recent studies find that the resistant pathogenic microorganisms mainly obtain antibiotics resistance genes by lateral gene transferring than mutagenesis as assumed before. Recent development in ecology and evolutionary biology of the environmental resistome, and the origin, evolution and the crossing dissemination of the resistant genes among the pathogenic bacteria and the environmental microbiome were reviewed. And also, the application of the high-through sequencing and metagenomics in the research of the environmental resistome were discussed.
The marine pharmacology research is getting stronger and stronger. Recent research has focused on the study of specific activity of marine natural products, and applied to new drugs for human. Phloroglucinol is a kind of important pharmaceutical intermediates. It is widely distributed in marine alga,especially in Phaeophyta. As a non-atropine non-papaverine class smooth muscle spasmolysis medicine,phloroglucinol primarily treat a variety of acute spasmodic pain. It is the first choosed drug in the treatment of gastrointestinal, genitourinary tract spasms and contractions during pregnancy, for its fast-acting, less side effects. Now, phloroglucinol is mainly produced by chemical synthesis. But the chemical method has many disadvantages, so biological synthesis become a potential method and research hot spot to produce phloroglucinol. The mechanisms and the methods of phloroglucinol biosynthesis are reviewed. In addition, it also sums up the main applications of phloroglucinol.
