miRNA (microRNA) is a kind of small non-coding RNAs that generally exist in animals, plants, and other species. The study shows that there are miRNAs in protozoa and viruses. miRNAs play crucial roles in many biogenesis, for example, biological growth, development, cell apoptosis, neurological disorders, cancer and other aspects. miRNAs can also be used as biomarkers in the diagnosis and treatment of diseases such as cancer. miRDOA (miRNA Database and Online Analysis) is designed to retrieve and analyze sequencing data and expression profiles of miRNAs. It provides browsing and searching function for miRNAs, targetor sequences. It also provides online analysis of High-throughputsequencing data, target prediction,expression profiles analysis and online BLAST. miRDOA will be updated over time and is free available for non-commercial use atmirdoa.biocloud.org.cn.
Discoveries from cancer genome sequencing have great potential application value for cancer prevention, diagnostics, prognostics, treatment and basic biology. Given the diversity of downstream applications, cancer genome-sequencing studies need to be designed to best fulfill specific aims. At the same time, knowledge of second-generation cancer genome-sequencing study design also facilitates assessment of the validity and importance of the rapidly growing number of published studies. Here, we focus on the practical application of second-generation sequencing technology to cancer genomics, and discuss how the study design and method could be adjusted to better achieve the purpose of specific aims.
It has been widely proved that the emergence and transmission of H7N9 avian influenza in China 2013 is closely associated with the live poultry trading all over the mainland China. By analyzing the poultry trading information in mainland China from websites using big data analysis technology, we could perform the work of H7N9 outbreak tracing,transmission analysis and trend prediction. In current study, we obtained the nucleotide sequences of hemagglutinin gene of H7N9 avian influenza virus isolates collected before 2013 from the Influenza Research Database firstly, and then conducted phylogenetic analysis using maximum likelihood method by RAxML software. A phylogenetic tree was constructed and then based on the phylogenetic relationship and isolate background including sources, time and locations of collection, we built several transmission routes of H7N9 between provinces and cities during the outbreak emerged in China in the first half of 2013. The results from phylogenetic analysis were compared with the ones from big data analysis to modify the transmission model which has been built by the big data method in our previous research. It revealed that the phylogenetictree could provide more accurate transmission information to trace the real route which could be used to complete and modify the model from big data, but the connection and transmission model from big data analysis could provide nearly accurate information from larger areas in time, which is meaningful in facing the H7N9 outbreak and could provide valuable basis for setting up and carrying out health policy and measures of disease control and prevention.
Simple sequence repeats (SSRs) or microsatellites have been successfully used for various genetic and evolutionary studies in eukaryotic, prokaryotic and viral systems, but information regarding SSRs in bacteriophages is limited by lack of studies. We made a comprehensive analysis of microsatellites and compound microsatellites (composed of two or more microsatellites residing directly adjacent to each other) in 60 Caudovirales genomes, and a total of 11 874 microsatellites and 449 compound microsatellites were observed in these genomes. In general, the count of SSRs is proportional to genome size by Pearson linear correlation analysis (ρ=0.899,P<0.01). Microsatellites in reference sequences are lower than that in random sequences, and it was beyond our expectation. The primarily reason was that reference sequences contain fewer mononucleotide and dinucleotide repeats. A/T and AT/TA were predominant in mononucleotide and dinucleotide repeats in most sequences, so GC content in mononucleotide repeats was notably lower than that in corresponding genome; by contrast, GC content has no significant difference between reference sequences and microsatellites of dinucletide and trinucleotide repeats. These results in Caudovirales are more or less different from other organisms' genomes. Our study might be helpful in understanding the distribution, evolution and biological function of microsatellites in Caudovirales.
The regulation of DNA replication initiation sites has a guarantee for DNA replication and cell division. Nucleosome positioning is an important factor for the regulation of DNA replication initiation sites. But it is not clear that how nucleosome positioning regulates DNA replication initiation. It has important biological significance to understand the mechanism of DNA replication initiation. In the study, ten autonomously replicating sequences(ARS) of Saccharomyces cerevisiae YPH499 Ⅲ chromosome were divided into efficient and inactivity groups. These aim sequences were amplificated by PCR, then recycled and purified using TIANgel Maxi Purification kit. A gradient salt dialysis method was employed to assemble nucleosome in vitro, then nucleosome depended on the purpose sequence were analyzed with Biotin labeling method. The ability of forming nucleosome was analyzed with Image J software.The result showed that nucleosome assembly strength which was analysised by Image J software is respectively 0.706, 1.395, 2.989, 0, 0.031, 0, 0, 0.714, 0 and 0 from ARS302 to ARS315. In other words, nucleosome assembly strength was as follows:ARS304>ARS303>ARS313>ARS302>ARS306>ARS314,ARS305,ARS307,ARS309,ARS315. The results indicated the inactive ARS were more likely to form nucleosome than the efficient ARS; Replication initiation sites often show anucleosome-depleted region.
To improve the efficiency of electricity production of MFC, it is necessary to understand the principles of extracellular electron transfer(EET) of Shewanella oneidensis MR-1,for example, the EET pathways under specific conditions(different grow conditions or different terminal electron acceptor and so on).41 c-type cytochromes of Shewanella oneidensis MR-1 were obtained from literatures and 50 related proteins were downloaded in STRING, then the coding genes of these proteins in NCBI. As the GRN of Shewanella oneidensis MR-1 had been reconstructed before, and 3367 transcription relationships were discovered, in which the transcription relationships between c-type cytochromes related genes were identified.Using R platform, we built the gene regulatory networks and explored the possible pathways to clarify the principles of EET, the bacterial growth conditions were controlled by using different gene expression datasets. At last, three specific EET pathways were proposed while analyzing GSE7973,GSE15334 and GSE25821.
Recently there were increasing reports about vaccines based on whole recombinant Saccharomyces cerevisiae. Mycobacterium tuberculosis (Mtb) protective antigens ESAT6 and Ag85B against tuberculosis were selected to be expressed in S. cerevisiae with pHR expression system. Two yeasts producing fusion proteins ESTA6-Ag85B (EA) and IFN-γ-ESAT6-Ag85B(IEA)respectively were constructed and the immune responses elicited by the recombinant yeasts were investigated in mice. Injection of mice subcutaneously with the recombinant yeasts induced Ag85B-specific IgG in high level and Th1 immune responses associated with IFN-γ and IL-2 secretion and no IL-4 production. Compared to BCG, Yeast-IEA vaccination stimulated significantly stronger immune response. It was confirmed that yeast could activated dendritic cells maturation with upregulation of co-stimulatory molecules and MHC molecules. The results suggest that the whole recombinant yeast (Yeast-IEA) may be an attractive candidate of vaccines against tuberculosis.
Objective: To express and purify herpes simplex virus 1 (HSV-1) glycoprotein D (gD) in Escherichia coli, and analyze its immune activity. Methods: The gD gene was amplified by PCR from HSV-1 genome and then cloned into the prokaryotic expression vector pET-28b. Then the recombinant vector pET28b-gD was transformed into E. coli cells, and the recombinant gD protein was induced by Isopropyl-β-D-1-Thiogalactopyrano-side (IPTG). The influences of induction conditions including IPTG concentration, period and temperature on the gD expression were analyzed. The inclusion body was lysed by the Guanidine HCl, and purified by Ni-affinity chromatography,and refolded by dialysis. And then the immune activity of gD was detected by Western blotting and ELISA. Result: Restriction endonuclease analysis and DNA sequencing confirmed that HSV-1 gD gene had been cloned into pET-28b. After analysis by SDS-PAGE, the gD protein was expressed in inclusion bodies form and the size of gD protein was 40 kDa. The best expression condition for gD protein involved induction with 0.5mM IPTG for 8 hours at 37℃. The total gD protein purified with Ni-affinity chromatography was 3.1 mg/L, and the total gD protein was approximately 1.3mg/l after refolding, so the refolding yield of the gD protein was 41.37%. Western blot and ELISA analyses showed that the gD possessed immune activity. Conclusions: HSV-1 gD protein with immune activity was expressed and purified in E.coli,which lays a foundation for the preparation of HSV diagnosis reagent and prophylactic vaccine.
Using genetic recombinant, prokaryotic expression, purification by Chitin-Beads column and HPLC, and identification by mass spectrometry technologies, a new VPAC2-specific agonist RD with the function of anti-type 2 diabetes was prepared, and its molecular mechanism of the effectively promoting insulin signal transduction in treating diabetes was studied. The results show that the molecular weight of the prepared RD is 3.785kDa. and its purity is 96%. Treating normal and insulin resistant 3T3-L1 adipocytes (IR model cells) with recombinant RD,the 1μmol/L and 5μmol/L RD treated normal 3T3-L1 adipocytes groups can significantly promote the expression of the IRS-1 protein, with an expression increase of 36% and 42% respectively. After the IR model cells were treated with the 1μmol/L and 5μmol/L RD, the IRS-1 expressions were increased by 55% and 63% respectively. After the IR model cells were treated with 1,5 and 10μmol/L RD, the pIRS-1(Ser307) expressions were decreased by 5.9%, 10.7% and 32.7% respectively. 5μmol/L and 10μmol/L RD treated IR model cells groups can decrease the expression of the IRS-2 protein by 12.8% and 40.6% respectively, and 1,5 and 10μmol/L MHDBAY treated IR model cells groups, the expressions of the pIRS-2 protein were decreased by 35.1%, 40.8% and 48.5% respectively. The expressions of the Akt protein in the IR model cells in 5μM and 10μM MHDBAY treated group were significantly increased, with an expression increase of 74% and 77% respectively. In IR model cells treated with 1, 5, 10μM RD, the phosphorylation levels of Akt Ser473 were decreased by,33.9%,64.0% and 71.1% respectively, and the phosporylation levels of Akt Thr308 were increased by 13.5%,78.6% and 83.3% respectively. This study established the technical preparation of the recombinant VPAC2 receptor agonist RD, and its biological effects in cell level were detected (significantly promote IRS-1 expression in normal 3T3-L1 adipocytes and IR model cell, decrease pIRS1 (Ser307), IRS-2, pIRS2 expression in IR cell model, and promote Akt expression and Akt Thr308 phosphorylation level, etc.). That may provide the experimental basis for illuminating molecular mechanism of RD in treating diabetes and its pharmaceutical research and development.
A frdB defection strain,SPUEC103(△frdB) was constructed,by using λ-RED homologous recombination technology with QLUEC001(produced succinate) as the parent strain. The fumarate accumulated by decreasing the flux of conversion of fumarate to succinate.frdB The fermentation results showed that the mutants of Escherichia coli deficient in frdB showed slower growth,together with exploiting less glucose.At the same time,the defection of frdB could change the yields of succinate and fumarate,the yield of succinate and fumarate was the highest in LB media supplemented with 30 g/L glucosethe,the yield of succinate of dual-phase fermentation decreased from 24.6% to 15.4%.The portion of fumarate and malate in SPUEC103 increased,the final concentration of the fumarate and malate was 0.182±0.002 g/L and 0.023±0.002 g/L.And the concentration of pyruvate and acetate decreased from 1.87±0.02 g/L and 0.012±0.002 g/L to 2.36±0.03 g/L and 0.862±0.012 g/L,respectively.
Bacillus can produce a variety of physiologically active substances and has broad application potential in environmental remediation, biological control, oil recovery and other fields. Bacillus mojavensis JF-2 and B. amyloliquefaciens BQ-6 are high-yielding strains of lipopeptide, screened from oil field, but their practical application in microbial enhanced oil recovery (MEOR) is restricted by oxygen concentration, salt concentration and pH. It is a simple and efficient approach to transform microbial metabolic functions by protoplast fusion. In this research, the above two Bacillus strains were taken as the research objects, and a L9(34) orthogonal experiment was conducted to explore the optimal conditions for protoplast preparation and regeneration. In addition, engineered bacteria were constructed by inactivated protoplast fusion. The results showed that cell age, lysozyme concentration and enzymolysis time significantly affected the rate of Bacillus protoplast preparation and regeneration rates (P<0.05), and the preparation rate was improved markedly when the Bacillus cells were repeatedly washed with sterile normal saline before enzymolysis. The optimum conditions for protoplast formation and regeneration of both Bacillus strains were: 7-hour cell age, 2.5 mg/ml lysozyme concentration, 30-minute enzymolysis, and 42 ℃ of lysozyme temperature. Fusant HY-4, salt tolerance of 15% and pH range of 4.0 to 9.0 for lipopeptide production, was capable of producing metabolic lipopeptide under both aerobic and anaerobic conditions, and it exhibited a rapid anaerobic growth (dry cell weight>1.6 g/L). In summary, fusant HY-4 has a great application potential, and this research laid a foundation for Bacillus genetic breeding methodology, providing a reference for breeding oil-flooding microorganisms.
Scenedesmus acuminatus was a new isolated freshwater green microalga cultured in modified BG-11 medium. In order to improve the rapid accumulation of the lipids, the initial NaNO3 concentration reduced to one third and one fifth of the original NaNO3 concentration in the BG-11 medium, 6.0mmol/L and 3.6mmol/L, respectively. It was large volume cultured in a new-designed internally installed tiepiece flat panel photobioreactor. To analyze the growth and oil accumulation pattern of S.acuminatus mass cultures, the biomass, total lipids content, lipid compositions, and fatty acids profiles in different phase were investigate. When the initial NaNO3 concentration was 6.0mmol/L, the biomass(6.27g/L)was higher than the biomass(5.30g/L) of 3.6mmol/L treatment. While, the highest lipids content of 56.6% of dry weight was occurred at 3.6mmol/L treatment. The total lipids content was fractionated by solid phase extraction (SPE) into three broad classes: neutral lipid (NL), glycolipid (GL) and phospholipid (PL). The content of neutral lipid increased along with the culture time, and it reached to 90.9% and 92.0% of the total lipids, 47.5% and 51.4% of dry weight when the initial NaNO3 concentration was 6.0mmol/L and 3.6 mmol/L, respectively. The major fatty acids of S.acuminatus were C16:0, C16:1, C18:0, C18:1, C18:2,and C18:3,which together accounted for 89.9%~96.2% of the total fatty acids content and 12.5%~50.7% of dry weight. The volumetric productivity of total lipids, neutral lipids and total fatty acids of S.acuminatus were 0.18g/L·d, 0.16 g/L·d and 0.15 g/L·d when the initial NaNO3 concentration was 6.0mmol/L and 0.16g/L·d, 0.15g/L·d and 0.15g/L·d when the initial NaNO3 concentration was 3.6 mmol/L, respectively. The results showed that S.acuminatus was a hyper-oil producing strain that is easy to large-scale cultivation and its profiles of fatty acids is suitable for biodiesel production.
Photosynthetic organisms synthesize the amphipathic antioxidant called vitamin E which are essential components of the human diet. Tocophero1 and tocotrieno1 comprise the vitamin E class in plants. Besides the antioxidant properties, the tocotrieno1 forms of natural vitamin E also help to lower cholesterol, prevent diabetes, promote bone resorption and reduce the risk of cancer and neurological diseases. Thus vitamin E is widely used in pharmaceutical, food and cosmetic industries. In this review, current knowledge on vitamin E biosynthesis pathway and related enzymes was described. Moreover, recent studies on genetic engineering to enhance and alter vitamin E content and composition in plants were summarized. Co-expression of multiple genes in vitamin E biosynthesis pathway and plastid transformation by using synthetic multigene operons have provided new strategies to increase vitamin E production in plants.
Gene recombination system is required to meet the rapid development of plant gene engineering. With the advantages of highprecision and efficiency,site-specific recombination systems havebeen widely used in the field of genetic engineering. The mechanism, advantages and disadvantages, and application of three site-specific recombination systems are summarized in the article, which are expected to contribute to the researches on plant transgenic engineering.Meanwhile, the current research hotspot for gene editing technology,the CRISPR-Cas system is introduced briefly in this paper.
Photosynthetic bacteria were concerned in environmental governance by its non-toxic, breeding fast, strong ability to adapt, easy artificial cultivation etc. The domestic and foreign research demonstrated that some photosynthetic bacteria played a key role in aquaculture (such as purifying water quality, making as a feed additive, et al) and in life and industrial heavy polluted water treatment. However, there were few reports about the role of photosynthetic bacteria in landscape slightly polluted water governance.Our researches indicated that Rhodopseudomonas palustris could effectively remove the ammonium nitrogen in the landscape water, and the highest removal rate can reach 95%, which implied that photosynthetic bacteria could effectively governance landscape water pollution. To provide reference for further studies of the functions of photosynthetic bacteria in landscape water, classification of photosynthetic bacteria, nitrogen and phosphorus removal principle and the application of photosynthetic bacteria in the treatment of organic wastewater, heavy metal wastewater as well as aquaculture wastewater were reviewed, and its developmental potential in landscape water pollution treatment was proposed.
The products of tissue engineering include artificial skin, blood vessel, cartilage, bone, cornea, cardiac valves, trachea, tendon, ligament, nerve, muscle, bone marrow, genital tract, urethral canal, intestine, breast, liver, kidney, pancreas, heart, bladder and hand, etc. Most of these products are in a stage of laboratory investigation exploration; less products are carrying out clinic test or ratified clinic application. The authorized tissue engineering products mainly include skin products, cartilage and bone products, cardiovascular products, nervous system products and artificial organs, etc., which are usually applied in clinic. More and more tissue engineering products will be emerged in future, and the clinic use of tissue engineering products will be more and more extensive.
