Objective : To investigate the decrease of Numb protein expression in triple-negative breast cancer patients and the effect of Numb protein on the p53 protein levels in triple-negative breast cancer and its regulation mechanism, and to further study the correlation between the decrease of Numb protein and the occurrence and development of triple-negative breast cancer, so as to provide a potential new therapeutic target for triple-negative breast cancer without effective treatment.Methods: 40 cases of triple negative breast cancer were taken from Clinical pathology diagnosis centre of Medical University of Chongqing. The expression of Numb protein in triple negative breast cancer patients was detected by immunohistochemistry. MCF-10A cell line and MDA-MB-231 cell line were all ATCC-derived. The mRNA and protein levels of Numb, HDM2 and p53 were compared by qPCR and Western blot. Numb was re-expressed in MDA-MB-231 cells by plasmid transfection. The expression of Numb, HDM2 and p53 was verified by qPCR and Western blot.Results: After transfection with NUMB-EGFP, the mRNA and protein levels of Numb in MDA-MB-231 cells were significantly increased, HDM2 did not change evidently, and p53 did not change significantly at the mRNA levels, but increased remarkably at the protein levels. The increased of Numb protein in NUMB-EGFP-transfected cells can regulate the p53 levels at post-transcriptional level, resulting in a significant increase of p53 protein levels.Conclusion: The expression of Numb protein in triple-negative breast cancer patients decreased by 55%, and Numb protein can regulate the level of p53 protein in triple-negative breast cancer cell line MDA-MB-231. The level of Numb protein is positively correlated with the level of p53 protein.
Objective: A strain of Pichia pastoris producing fusaruside was constructed to solve the source problem of fusaruside, a selective immunosuppressive molecule.Method: Two related biosynthetic genes coding delta 3(E)-sphingolipid desaturase [Δ3(E)-SD] and delta 10(E)-sphingolipid desaturase [Δ10(E)-SD] were amplified from fungus Fusarium graminearum PH-1. 2A peptide based strategy was used to construct the co-expression vector. Then the 2A polyprotein construct was transformed into Pichia pastoris GS115 for induction. Finally, P. pastoris cells were extracted with methanol and dichloromethane, and the extract was detected by high performance liquid chromatography mass spectrometer (HPLC-MS).Result: The two desaturases were successfully co-expressed in P. pastoris. SDS-PAGE showed that the molecular weight of Δ3(E)-SD and Δ10(E)-SD were about 48kDa and 65kDa, respectively. HPLC-MS indicated that fusaruside could be produced by the recombinant yeast.Conclusion: Compared with fusarium which producing fusaruside, the engineered yeast had shorter fermentation time and higher yield, laying a foundation for further development and application of fusaruside.
6-Hydroxynicotinic acid (6HNA) 3-monooxygenase (NicC) is the key enzyme for nicotinic degradation in Pseudomonas putida KT2440. NicC can catalyze the hydroxylation of pyridine ring to promote the ring cleavage reaction of pyridine ring. The expression level of NicC was enhanced by replace the rare codon in the N-terminal of NicC, and then the His-tagged NicC was purified to homogeneity. The optimal temperature reaction range of NicC is from 30℃ to 40℃, and the optimal reaction pH is 8.0. The Cd 2+ could significantly inhibit the activity of NicC. The apparent Km and Vmax values of the purified NicC for 6HNA were 51.8μmol/L and 14.1U/mg, respectively, and those for NADH were 15.0μmol/L and 10.79U/mg, respectively. According to the HPLC and LC-MS analysis, NicC could catalyzes 6HNA to form 2,5-DHP and formic acid, and it could also transform 4-hydroxybenzoic acid to form hydroquinone. Isotope labeling experiments proved that the oxygen atom incorporated into 2,5-DHP is from dioxygen. The study will provide useful information for the microbial degradation of pyridinic compounds.
A cellobiose 2-epimerase gene ce derived from Dictyoglomus thermophilum was synthesized and introduced into the expression vector pBSuL3 to construct a recombinant plasmid pBSuL3-ce and then transformed it into Bacillus subtilis. The intracellular activity of cellobiose epimerase reached 7.5U/ml after cultivated in TB culture for 48h. The enzymatic properties showed that the optimum pH of the enzyme was 8.5;the optimum temperature was 85℃, and the half-life of 85℃was 120min. In order to reduce the cost of fermentation, the fermentation medium was optimized.When 35g/L soybean meal was used as nitrogen source and 5g/L glycerin was used as carbon source, the intracellular activity reached 12.3U/ml. Then, according to the optimized condition of the shake flask, the culture was expanded in the 3L fermenter, and the intracellular activity of cellobiose epimerase reached 56U/ml, which was 8-fold higher than that of the shake flask culture.The lactulose was prepared by using the enzyme obtained by fermentation. When the lactose concentration was 400g/L, the reaction temperature was 85℃, the initial pH was 8.5, the enzyme amount was 20U/ml, the enzyme converted lactose to 51% lactulose.
Straw plastic composite (SPC) is a novel wood - plastic material in which wood fibers are replaced by straw fibers.Wheat straw / rubber biomass imitation rattan from wheat straw, rubber and low density polyethylene( LDPE) was prepared. The changes of mechanical properties and microstructures were investigated in the period of thermal-oxidative aging at 100℃ for 60d. The results showed that the addition of wheat straw fiber will reduce the mechanical properties while the rubber can enhance the elongation break. During the aging process, cracks appearing on the surface of rattan cut down mechanical properties. And the losing of functional groups between the surface of straw fiber and PE caused the reduction of combining ability. In combination with the kinetic model, the degradation occurred rapidly in the beginning of the 15 days with the aging coefficient up to 70% and kept a low rate in the rest of the experiment. Furthermore, the addition of rubber could slow down the aging rate of rattan.
Objective: To generate a JAK2 knockout K562 cell line by CRISPR/Cas9 gene editing system.Methods: Using CRISPR online design tool, sgRNA was designed targeted to JAK2 gene and used to construct the co-expression plasmid of Cas9-sgRNA. Lentivirus was packaged by the second-generation lentivirus packaging system and used to transduce K562 cells, the genomic DNA of cell pool was extracted, and the gene editing activity was detected by Sanger sequencing and TA cloning. The candidate edited cells were inoculated into 96-well plate by infinite dilution method. Then the cells were expanded to extract genomic DNA. The JAK2 sequence was identified by Sanger sequencing and TA cloning.Results: LentiCRISPRv2-sgRNA3-1 plasmid containing the gene editing tools for koncking out JAK2 was constructed. The optimal dose of lentivirus with low cytotoxicity and high transduction efficiency was obtained. The JAK2 gene knockout K562 cell line (K562-JAK2 -/-) was successfully generated.Conclusion: Lentivirus transduction-based CRISPR/Cas9 system was successfully used to generate JAK2 gene homozygous knockout K562 cell line, providing a cell model to study the importance of JAK2 gene in the field of chronic myeloid leukemia. Besides, the lentivirus transduction-based CRISPR/Cas9 protocol reported here lays a foundation for constructing other gene knockout K562 cell lines to study of hematopoietic differentiation mechanism.
Pichia pastoris is one of the most widely used heterologous expression host in the synthesis of human insulin precursors both in laboratory research and industrial production. Currently, the induction fermentation production of insulin precursors is carried out mainly by using the naturally methanol-inducible AOX1 promoter and utilizing methanol as the sole carbon source. High oxygen consumption, heat release and complicated feeding process control caused by methanol catabolism usually bring difficulties to industrial scale-up and limited application for protein expression by high cell densities fermentation in Pichia pastoris. A synthetic efficient constitutive transcriptional signal amplification device of CSAD_5 was constructed to drive insulin precursor gene expression based on the transcriptional regulation design of the AOX1 promoter in our previous work. In order to reduce the oxygen consumption, heat generation and bioprocess regulation during fermentation process, a glucose supported fermentation process is established. Through increasing module-specific antibiotic stress to optimize the biosynthetic gene dosages, a recombinant Pichia pastoris strain producing 1.85g/L insulin precursor was screened in 108h in a 5-L bioreactor, which is the highest productivity to produce human insulin precursor by utilizing glucose as a carbon source according to reports. Our findings provide a new alternative choice to the high-yield production of insulin precursor in industrial application and reveal the potential of Pichia pastoris to produce a wide range of compounds.
Microalgal oil is an important functional oil, but also one of the major sources for bio-diesel production. In order to solve the contradiction of algae growth and lipid accumulation, the Chlorella vulgaris were co-cultured with azotobacter at different initial ratio under nitrogen source deficiency conditions, and the impact of consortium system growth, lipid accumulation based on the dry weight, lipid content, and fatty acids composition in the incubation period were evaluated. The results showed that the biomass and lipid content of algae were significantly higher than that of the pure culture of axenic C.vulgaris when C. vulgaris and B2.3 strains were inoculated at the ratio of 70∶1. The biomass, lipid content, lipid productivity, neutral lipid content and neutral lipid productivity of algae in the co-cultures were 1.68g/L, 45.2%, 75.94mg/(L·d), 23.0% and 38.65mg/(L·d), respectively. And the biomass concentration and lipid content in the co-culture system were 66.3% and 47.7% higher than that of the axenic pure algal cultures, respectively. Furthermore, the consortium system significantly increased the proportion of C18∶1 fatty acids. It was indicated that the co-cultivation of algae-bacteria system can effectively contribute to the quality and quantity of microalgal bio-oil, and has great potential for production of bio-diesel.
Trehalose was prepared from rice starch by multiple enzymes reaction. The optimum conditions for the production of trehalose were determined under laboratory conditions: 15% (m/V) rice starch as substrate, catalytic temperature 45℃, pH6.0, DE 16,α/β-CGTase 1.4U/ml, catalytic 28h and period 12h. The conversion of trehalose increased from 50% to 73% by the innovated method. When the substrate concentration was 25%, trehalose yield reached 182.5g/L. Then high concentration trehalose was separated and extracted. The effects of decolorization,resin separation, concentration and crystallization of activated carbon on trehalose extraction were investigated.
Covalent binding is one important immobilization method of industrial enzyme, which uses stable covalent bonds to immobilize industrial enzymes, creates multi-point covalent connections between carrier and enzyme, and prepares immobilized enzymes with good stability and possesses practical application value. Marine Candida rugosa lipase was immobilized using amino carrier through covalent binding method and relative inexpensive glutaraldehyde was used as the crosslinking reagent. By using single factor and orthogonal experiment, the optimal immobilization conditions were determined as follows:25℃, pH5.0,0.1% glutaraldehyde,0.25g carrier, crosslinked 0.5h,immobilization time 1h, enzyme loading 800U, with the final obtained enzyme activity being 83.01U/g. Compared with free enzyme, the optimal pH of immobilized lipase was shifted to alkaline direction, the optimal reaction temperature was increased by 10℃, the thermal stability and acid-alkali stability of immobilized enzyme were better than that of free enzyme, and the reusability and storage stability were better than that of free enzyme. Meanwhile, crosslinking agent was found to be an important factor in the preparation of immobilized lipase. Thus it is of great significance to explore new crosslinking agent to improve immobilization effect. A good foundation for the immobilization technique and industrial application of marine candida rugosa lipase were laid.
Chemotherapy resistance is one of the major challenges in the treatment of lung cancer. Chemotherapy resistance results in therapy failure and missing the opportunity of optimal therapy for some patients. Therefore, it is very important to study the mechanisms of chemotherapy resistance for lung cancer treatments. MicroRNA (miRNAs), small molecules of RNA, are involved in the regulation of various biological processes and play important roles in the therapy resistance of lung cancer. It’s shows that miRNAs regulate drug resistance by reducing the expression of multiple drug resistance-related genes or promoting cell escape apoptosis in cancer cells which treated with chemotherapeutic agents. However, the mechanism of microRNA-mediated drug resistance is not fully understood. Abnormal regulation of specific miRNAs may be associated with chemotherapy resistance to a variety of cancers, there by regulating the sensitivity of cancer cells to drugs in lung cancer. Here will systematically elaborate provide up-to-date understanding in the roles and mechanisms of miRNAs in chemotherapy resistance of lung cancer.
With the in-depth development of proteomics related technologies, the study of pathogen invasion and host interaction is becoming more and more in-depth, and the high-throughput and large-scale analysis of protein interaction and protein expression patterns is possible. Through the systematic analysis and identification of proteins, it has a high reference value for the occurrence and development of diseases. Antibody chip analysis of proteins with the advantages of micro-integration, large-scale and high-pass quantification, has been widely used in the field of biomedicine. The current research progress of antibody chip technology and its application in parasite research are reviewed. The development process of parasites has its unique life history, and the expression of parasite proteins and excretory and secretory products at different developmental stages are very different. antibody chip technology is applied to proteomics research of parasites to screen specific marker antigens. It is of great significance to explore the growth and development of parasites, colonization invasion, immune escape and immunosuppression, so as to lay a foundation for the early diagnosis and treatment of parasitic diseases and the search for new drug targets. At the same time, it provides a new idea and method for the further study of parasite proteomics.
Xylan is abundant in crops and agricultural and forestry residues. With the increasing emphasis on energy resources, more attention has been paid to the application and research of xylan. Xylanase is a class of hydrolases that can degrade xylan into xylooligosaccharides and xylose. Several improvements were made over the year to achieve high yield and high enzyme activity of xylanase.The research progress of heterologous expression of xylanase were reviewed.
Streptonigrin (STN) is an anti-tumor antibiotic with a unique aminoquinolinium structure produced by Streptomyces flocculus, which has good control effect and broad application prospects in cancer treatment. However, the clinical application of STN has been limited because of its relatively significant myelosuppression side effect.The anti-tumor mechanism, structural features and existing knowledge related to the biosynthesis of streptonigrin and its analogs, including research status and prospects of reducing toxic side effects have been summarized, and providing a scientific reference for the development of low or non-toxic streptonigrin and its analogues.
The third large-scale merger and reorganization of multinational seed companies have formed a new international seed pattern,intellectual property protection is one of the biggest drivers of cross-border seed mergers and acquisitions. Studying the accumulation and reorganization of the core intellectual property rights formed by the merger of multinational seed companies, it is possible to further examine the changing trend of the new pattern of international seed industry competition. One of the trends, the United States, Japan and Europe are still the leaders and monopolists of global breeding research and development innovation activities, large-scale mergers and acquisitions, achieving the integration of technology and resources, and promoting the concentration of international seed industry, 42.75% of patent applications of the global seed industry is concentrated in the United States, Japan and the European Union, and holding 64.68% of the global DNA recombination technology patents. Trend 2, intellectual property has become a powerful weapon for multinational seed companies to maintain market competitive advantage, and multinational seed companies is the owner of core technology patents. Bayer/Monsanto, China Chemical/Syngenta and DuPont Pioneer/Dow’s seed patent applications for global breeding is nearly 14%, and more than 81% of the global crop transformants are controlled by multinational seed companies. Research and analysis on the changing trend of the new pattern of international seed industry competition can provide valuable policy advice for China’s seed industry mergers and acquisitions.
