Objective:To explore the effect of glutamate dehydrogenase deletion on biofilm formation, virulence and extracellular proteins expression of Listeria monocytogenes.Methods: The effect of glutamate dehydrogenase deletion on the formation of L.monocytogenes biofilms was examined using a microtiter plate method. The hemolytic activity, the semi-lethal dose of Helicoverpa armigera (Hübner)in wild-type, GDH-deficient and revertant strains were compared.The differentially expressed extracellular proteins between the wild strain and GDH-deficient strain were identified and analyzed by iTRAQ technology. Results: Compared with wild strains, the amount of biofilm formed by the GDH-deficient strains was significantly decreased (P≤0.01), as well as the hemolytic activity, but its semi-lethal dose on Helicoverpa armigera (Hübner) was increased 1.6 fold. The functions of all sixty-two differential expressed extracellular proteins identified by iTRAQ were cataloged into 16 functional groups, including carbohydrate transport and metabolism, energy synthesis and transport, transcription, cell membrane protein related to cell wall and so on. Among them, the most dominant groups including 24 proteins, accounting for 38.71% of total differential expressed proteins, are related to carbon and nitrogen metabolism as well as energy transport, suggesting that glutamate dehydrogenase is a key enzyme in carbon and nitrogen metabolism as well as energy synthesis and transport for L.monocytogenes. The slow growth and reduced biomass of EGDeΔgdhA compared to EGDe in minimal essential medium were in agreement well with these proteomic results. In addition, the expressions of three proteins from EGDeΔgdhA associated with bacterial adhesion and biofilm formation were also significantly decreased.Conclusion: Deletion of glutamate dehydrogenase reduces bacterial biofilm formation and virulence, affects significantly the extracellular protein expression in L.monocytogenes.
Mortierella alpina is a filamentous fungus with a high polyunsaturated fatty acids content,but its sources of NADPH needed for lipid synthesis are not been studied completely. The binary vector pBIG2-ura5s-MTHFD1 was constructed for the transformation of the methylenetetrahydrofolate dehydrogenase(MTHFD1)into the M.alpina (uracil auxotrophic) strain by Agrobacterium tumefaciens-mediated transformation (ATMT). PCR analysis identified the presence of the MTHFD1 overexpression cassette in the genome, and qPCR analysis showed that the transcript levels of the MTHFD1 gene in MTHFD1 overexpression strain (MA-MTHFD1) were significantly increased compared to controls. The results showed that MTHFD1 overexpression influenced lipid synthesis significantly. In comparsion to prototrophic M. alpina, the total fatty acid(TFA)level increased by about 40.13% and NADPH level simultaneously increased by 26.45% in MA-MTHFD1. Moreover, two key enzymes ME and IDH in NADPH synthesis were up-regulated. Based on the results, MTHFD1 plays an important role in NADPH generation during lipid synthesis in M. alpina. A foundation for uncovering mechanism of lipogenesis in M. alpina was established.
Chitosanase encoding gene of Bacillus subtilis 168 was optimized, synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed product reached 0.30mg/ml. The optimum pH and temperature of the expressed chitosanase was 5.6 and 55℃, respectively, and enzymatic activity reached 84.54U/ml. The chitosanase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition of these products were analyzed through utra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). The results showed that these hydrolysates contained at least 37 different kinds of chitooligosaccharides with degree of polymerization of 2-18 and different degree of deacetylation. In summary, chitooligosaccharides with low degree of deacetylation were prepared through Bacillus subtilis 168 chitosanase expressed in Pichia pastoris and its composition analyzed, which can provide a reference for the study of the relationship between the structure and function of chitooligosaccharides.
As an excellent species for the production of DHA, Schizochytrium sp. has two distinct pathways for producing DHA, fatty acid synthase pathway and polyketide synthase pathway, of which fatty acid synthase pathway played limited role in the DHA synthesis process due to its low activity of one or more of the enzymes, resulted in its only responsible for some short chain fatty acid synthesis. In order to enhance the ability of fatty acid synthase pathway for producing DHA in Schizochytrium sp. to improve the DHA content of its oil, the exogenous △12-desaturase expression plasmid with screening markers for Schizochytrium sp.ATCC20888 from the brown algae was constructed, then Schizochytrium sp. were transformed through electroporation. After long-term screening, two positive transformants were obtained by DNA and RNA identification. Their initial biomass measurements were 11.14% and 4.12% higher than the wild strains while the DHA content in oils and fats were 19.50% and 14.65% higher than the wild strains.
Waldenström’s macroglobulinemia (WM) is a rare, incurable lymphoplasmacytic lymphoma (LPL). Research has showed that MYD88 L265P is commonly detected in patients with WM (>90%). Therefore this mutation could be used as a biomarker for disease diagnosis and differentiation. Current detection methods used at this mutation are of low sensitivity, which limit the clinical utility for early diagnosis. Thus, there is an urgent need for a more sensitive method to fulfill the requirement of early diagnosis. By combining ARMS and clamping PCR together, a novel detection system with higher sensitivity was developed. The limit of detection of the new system was 10 2 copies and the sensitivity was 0.1%. A double-blind trial with 19 clinical samples was conducted and the results completely accorded with the sample information. This new technique has a huge potential in clinical practices, especially early diagnosis, due to its increased accuracy and sensitivity.
Objective: To establish an in vitro method of calcium channel Orai1. Methods: The purified orai1 protein was reconstituted into liposome membrane by the liposome reconstitution technology, and the recombinant efficiency and the structure of Orai1 protein on the liposome membrane were detected by sucrose density gradient centrifugation, and the release of calcium in the liposome was detected by calcium dye Fura-2.Results: The liposomes were successfully prepared and the GST-Orai1 fusion protein was purified in vitro. The sucrose density gradient centrifugation showed that the GST-Orai1 protein was successfully reconstituted into liposomes, and the Orai1 protein was located on the liposome membrane in the form of polymer. Calcium release experiments showed that calcium ions were loaded into the liposomes and could be used for functional study of Orai1 calcium channels.Conclusion: A new method of Orai1 was established to study its function and activation mechanism more directly and effectively.
Objective: To develop a simple, quick and sensitive method based on the LNA (locked nucleic acid) PCR for detection of rtA181V and rtN236T mutations associated with adefovir dipivoxil (ADV) resistance of hepatitis B virus (HBV). Method: Built wild strains and the mutant recombinant plasmids of ADV rt181 and rt236 by gene sequencing screening positive samples, then designed the specific primers and LNA fluorescent probes (rtA181V, rtN236T) to construct recombinant plasmid for standard real-time fluorescent PCR reaction system, and determined the feasibility and accuracy of the detection method through the parallel to gene sequencing to detect serum samples. Result: LNA-PCR assay could detect 10 2 copies/ml mutant template, with high specificity. Eighty-nine HBV DNA positive samples were from patients with ADV therapy over one year. LNA-PCR detected two (2.24%) rtA181V and rtN236T dual mutations, eight (8.98%) rtA181V mutations, five (5.61%) N236T mutations. Complete concordance between LNA-PCR and sequencing were observed with all samples. Conclusion: LNA-PCR test is a simple, fast, and sensitive method for monitoring ADV resistant mutations of HBV in patients with chronic hepatitis B.
Objective: To prepare the Ucp2 gene RNA probe labeled by digoxin, and was used to detect the early Ucp2 gene spatiotemporal expression in mouse embryos. Methods: The primers were designed and the total RNA was extracted from fetal mice nervous tissue. The Ucp2 gene fragment was obtained by RT-PCR and cloned into the pGEM -T vector. Then transcription template was obtained using Sp6, T7 and Ucp2 primers by PCR. Using Sp6 and T7 RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole embryo in situ hybridization. Results: The plasmid pGEMT-UCP2 was constructed. The digoxingenin-labeled antisense Ucp2 probe was effectively detected in mice Ed9.5 and Ed10.5, and the expression was not detected by the sense probe. Conclusion: The antisense Ucp2 probe can be used specifically and sensitively for study in situ hybridization and lay the foundation for further research of Ucp2 expression in the tissue of mouse embryo, especially in the nerve tissue.
(S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate [(S)-CHOH] is the key chiral intermediate of statins. Asymmetric reduction of tert-butyl-6-chloro-3,5- dioxohexanoate (CDOH) to (S)-CHOH catalyzed by alcohol dehydrogenases is a promising method. Nevertheless, the main problems is the low catalytic activity towards CDOH. First an alcohol dehydrogenase LkTADH (A94T/F147L/L199H/A202L) was further studied by reverse mutation and key sites (147,202) had been identified. MF147L-A202L was obtained, which demonstrated 1-fold improvement in specific activity over LkTADH. After applying saturation mutagenesis at these two sites, MF147I-A202L was obtained with 1.47-fold improvement in specific activity over LkTADH. The specific activity reached 10.17U/mg, which is the highest level as reported. Through dynamic analysis and molecular docking, the effect of mutation sites on enzyme activity was further analyzed.
Cancer chemotherapy and the patient’s life will be very convenient if oral administration of anti-cancer drugs can be achieved. For most anti-cancer drugs, their bioavailability and treatment efficiency will be decreased by oral administration, due to the gastrointestinal barrier. So it is important to find an effective drug carrier for oral administration of anticancer drugs. The emergence of nanotechnology, led to the development of nano-drugs. Therefor a great breakthrough has been achieved for oral administration of anti-cancer drugs. The research progress of different materials, including synthetic polymer materials, natural macromolecular materials and liposomes as oral anticancer drug carriers, including their in vivo or in vitro studies has been reviewed.
In recent years, magnetotactic bacteria and their biosynthetic magnetosomes have been recognized and have been used in biological and medical applications by people because of their good biosafety. Compared with synthetic magnetic nanoparticles, magnetosomes extracted from magnetotactic bacteria have biomembrane coated, high biocompatibility, uniform particle size and high magnetic. Because magnetotactic bacteria swam along magnetic field, they are also applied in biomedical applications. The characterization of magnetotactic bacterium and magnetosome are first descripted, then reviewed their apllications in biomedical in latest research progress.
Biobutanol has been attracting much attention as a clean fuel and chemical due to that the use of fossil fuels lead to aggravation of global warming and energy crisis. Escherichia coli is an ideal candidate for butanol production because it is easy to manipulate genetically. Butanol toxicity has been a bottleneck for industrial-scale biobutanol production, so the improvement in butanol tolerance is essential for high titer butanol production. Butanol destroyed the barrier and transport functions of cell membrane, and cell produces physiological response, which is similar to that of heat shock, osmotic stress, etc. Cell regulates transcription and translation to resist butanol stress. In the light of the above points, the butanol tolerance mechanism of E. coli and recent advances in development of butanol-tolerant strains by rational design strategy are summarized in this review. Nevertheless, the mechanism has not been yet fully elucidated, which limits the use of rational design strategy. There is also concern about the application of inverse metabolic engineering in this area, which means that the butanol-tolerant strains are obtained through directed evolution and the functional genes are further revealed. In addition, the progress on application of the latest strategies for improving butanol tolerance, such as combined strategy, chemical modification, and propose the potential key points for enhancing butanol tolerance of E. coli were reviewed.
In recent years, Pseudomonas aeruginosa has become one of the main pathogens of nosocomial infections, which is an opportunistic pathogen can cause acute or chronic multiple infections and is hard to be controlled by antibiotics. Studies have shown that the key to pathogenicity is the precise delivery of virulence factors to host cells by bacteria, while the secretory system plays an important role in this process. Among them, the recently discovered type VI secretion system (T6SS) is a type of secretion system that has drawn great attention from domestic and foreign,which plays an important role not only in the interaction between Pseudomonas aeruginosa and the host but also in the mechanism of promoting biofilm formation. Focusing on the studies of the structure, functions and regulatory mechanisms of Pseudomonas aeruginosa T6SS, a brief review was conducted to provide a new strategy for the treatment of patients with Pseudomonas aeruginosa infection.
The labeling of genetically modified(GM) products is closely related to the consumers’ right to know and the right to choose. Hence, the labeling policy has been a focus for concern. Since the promulgation of the first GM labeling policy in 1997, more than 70 countries and regions have carried out the labeling management of GM products. Since 2015, due to many factors including the development strategy of the GM industry, the public acceptance, the demand for the trade of agricultural products, and the government political policy, the United States, Korea, Russia, Ukraine and Japan have started to adjust the labeling policy or revise the implementation details. The changes are characterized by mandatory labeling requirements, expanded labeling scope, clearer labeling ways, and more standardized negative labeling (non-GMO labeling) requirements. Through the analysis of the international labeling policies and the recent trends, the suggestions on the management of labeling scope, labeling threshold and negative labeling in China are proposed.
Bio-based products (bioproducts) are materials,chemicals and energy derived from renewable resources (such as agricultural and forestry residues,organic waste). Because of its advantages of environment friendly,it has become an important way to be the sustainable development society. During these years,Developed countries including US and Europe have been deploying strategies to promote development of bio-based industry. By focusing on policy and financial support for bio-based research and innovation (R&I),European Commission (EC) has organized core funding framework and management system by BBI (bio-based industries) initiatives. As a typical case of R&I project in Europe,the proposal is put forward to carry out suggestion for bio-based R&I project implementation and other innovation directions with investigation and analysis of the bio-based project funding frameworks and budget allocations.
