To create a blood-brain barrier in vitro model by microfluidic chip technology, which were easy to control and close to microenvironment.
The microfluidic chips were composed of dual pool structure, separated by the polycarbonate membrane, and there were two sets of flow control system, mouse brain vascular endothelial cells and astrocytes were isolated and purified, the cells were identified by immunofluorescence. Then cells were added to the two pools according to the order, and infused at 1μl/min flow rate. The blood-brain barrier(BBB) model was constructed, and then identified and evaluated.
Two kinds of cells were obtained by isolation and purification, and the cells were identified by immunofluorescence and the purity was above 95%. After 3 days of co-culture, tight junctions began to develop, and the peak is on the fifth day, the ultrastructural observation showed that the mouse brain vascular endothelial cells formed tight junctions, the penetration experiments of fluorescein sodium and the measurement of TEER showed that barrier formation was good.
The blood-brain barrier model was constructed based on the microfluidic chip, It may become a new platform for drug screening, neural system basis and other studies.
To determine the effect of STAT3 on sorafenib resistance in hepatocarcinoma cell SK-Hep1, and the role of STAT3 gene silencing on enhancing the efficacy of sorafenib in liver cancer.
STAT3 gene was knocked down by short hairpin RNA based gene-silencing technology in SK-Hep1 cells. The cell proliferation and the sensitivity to sorafenib were detected by CCK8 assay. The expression levels of STAT3, p-STAT3(Y705),p-STAT3(S727)and its downstream proteins were detected by Western blot.
STAT3 knockdown cell line SK-Hep1-shSTAT3 was successfully constructed. In these cells, the level of STAT3 expression was decreased; the cell proliferation was significantly suppressed.Sorafenib treatment reduced the level of STAT3 phosphorylation and its downstream protein CyclinD1 and Mcl-1.Sorafenib resistance was reduced in STAT3 knockdown cells.
Silencing STAT3 by RNA interference can significantly suppress SK-Hep1 cell proliferation, and enhance the sensitivity of liver cancer cells to sorafenib.The data indicates that STAT3 knockdown might be a new way to improve the efficacy of sorafenibin liver cancer.
Protein E (PE), with two lysine residues at its C-terminus, is a plasminogen (Plg) receptor on the surface of nontypeable Haemophilus influenza (NTHi). NTHi can recruit Plg on the cell surface by PE and utilize host fibrinolytic system to achieve its immune invasion. Based on the high homology of Plg and Apolipoprotein(a) [Apo(a)] of Lipoprotein(a) [Lp(a)], Lp(a) was supposed to bind to PE.
The recombinant PE (rPE) and C-terminal lysine residues-deleted variant (rPEΔKK) were obtained by prokaryotic expression and further purified. Lp(a) was isolated and purified from human plasma by KBr density gradient centrifugation followed by Q SepharoseTM Fast Flow ion exchange chromatography. The interaction between rPE and Lp(a) was investigated by enzyme-linked immunosorbent assay(ELISA) and Pull down followed by Western blot.
The results indicated that rPE could bind to Lp(a) but not to LDL, and the interaction was significantly inhibited by EACA. The binding capacity of rPEΔKK to Lp(a) was obviously lower than that of rPE. In addition, Lp(a) could inhibit the binding of rPE to Plg slightly.
In overall, Lp(a) could bind to rPE and the C-terminal lysine residues of rPE and the lysine binding site(LBS) of Apo(a) was responsible for this interaction.
Early Indica rice variety “Jiayu 253” and “Zhongzu 3” were used as parents to breed a new variety “Zhongzao 39” through pedigree method and yield comparison trial. Natural disease nursery and artificial inoculation of mixed strains of blast for each segregation generation were conducted. The variety “Zhongzao 39” and its pedigree parents “Jiayu 253” and “Zhongzu 3” were further compared on yield related traits, photosynthetic characters, leaf area indexes and photosynthetic pigment contents. Results showed that total grain number per panicle and seed setting rate of Zhongzao 39 were higher than that of its pedigree parents, indicating its excellent sink. The highest net photosynthetic rate of Zhongzao 39 was demonstrated 20 days after the stage of full heading stage. Its leaf area index and photosynthetic pigment content were showed the most high as well. Therefore, Zhongzao 39 demonstrated elite photosynthetic physiological characters for high yield. Using high-yield pedigree parents in breeding and integration of elite traits during breeding contribute to the high-yield of Zhongzao 39. The super high yield advantage is obtained from source sink optimization, improvement of plant leaf morphology and performance of late-stage vigor super hybrid rice characters during rice breeding, which gives reference information for rice high-yield breeding.
Resveratrol is a member of stilbenoids with disease resistant activity for plants and exhibits a wide range of important biological and pharmacological properties for human which has received extensive attention in the fields of agriculture, medicine, foods, cosmetics and so on. Resveratrol synthase (RS) is an exclusive necessary enzyme in the pathway of resveratrol biosynthesis which determines the synthesis of resveratrol in plants. CiRS, a RS gene isolated from Caragana intermedia, was transferred into Arabidopsis. Total flavonoids experimental results showed that the total flavonoids content of wild type was significantly higher than that of the transgenic lines. HPLC method was used to analyze the resveratrol content in transgenic plants which maximum content was 335μg/g FW. Accumulation of malondialdehyde (MDA) after UV treatment in transgenic plants was significantly less than the wild type. DPPH free radical scavenging ability of transgenic plants extraction was higher than the wild type. Taken together, these results indicated that the antioxidant activity of transgenic plants was enhanced with the expression of CiRS gene which synthesise resveratrol with flavonoid substrates.
Cold-active CPC acylase plays an important role and has significant advantage in the biosynthesis of 7-ACA, thus developing cold-active CPC acylase has significant economic value. In order to obtain the CPC acylase with higher catalytic activity at low temperature, on the basis of previous research, the previously obtained CA IIIM was taken as a parent, confirmed its substrate binding region by the method of molecular docking, and found the key proline residues in the loop of CPC acylase substrate binding region by pyMOL. After analyzing, the selected prolines were mutated into glycines, respectively. The study of soluble expression was carried out in E. coli BL21(DE3) with pET32a plasmid, and soluble expression was achieved in the other mutants except P272G. Catalytic activity of P238G, P582G and P679G to CPC was 1.25, 1.04 and 1.38 U/mg respectively at 13℃, and there was a significant improvement compared with parental 0.85 U/mg. Moreover, the stability of parent and mutants was investigated, and there was no obvious difference between them. Afterwards, the low temperature biosynthesis of 7-ACA was carried out at 13℃, and the results showed that the conversion rate of CPC can reach 80% and above after 24h . The ideal results in cold adaptation improvement of CPC acylase were gained. So a solid foundation for further transformation and application was established, it provided a useful experience for the creating of other low-temperature enzymes.
To regulate the production of polyols by Trichosporonoides oedocephalis employing HOG1 inhibitors.
SB239063, SB202190 and SB203580 were added into the medium to conduct fermentation experiments, and the influence of three inhibitors on the fermentation was compared.
The experimental results showed SB239063 would decrease the ctGPD enzyme activity of Trichosporonoides oedocephalis, and enhance erythritol reductase(ER)enzyme activity at the same time.Furthermore, the Western blot analysis of HOG1 and phospho-HOG1demonstrated that SB239063 also inhibited the dephosphorylation of HOG1. After 120 h fermentation, the addition of 10μmol/L SB239063 decreased glycerol production by 20.57% and increased erythritol production by 31.16%.In other words, the conversion rate of glucose was increased by 24.73%.
SB239063 weakened the ability to produce glycerol of Trichosporonoides oedocephalis and therefore promoted erythritol yield.
According to the sequence of lysostaphin gene from Staphylococcus simulans and codon bias of Kluyveromyces lactis, the PCR primers were designed to amplify the fragment of lysostaphin gene. The fragment was inserted in pKLAC1, and transformed to K. lactis GG799. The K. lactis GG799/pKLAC1- Lys was cultivated to express Lys. A high expression strain (mu4#) were abtained by using powerful mutagen (N-methy1-N-nitro-N-nitrosoguanidine,NTG) on the recombinant and optimized the expression condition .The fermentation broth of mu4# was purified by Ni-NTA agarose and the enzyme characterization was studied. The result showed that the activity of Lys was approximately 5.2 times (8 000U/L) higher in the mutation. The optimal inoculum dose of the mutant (mu4#) was 40g/L; Galactose and NH4NO3 (20g/L) were added in every 24 hours, Lys exhibited optimal expression at pH 7.0~7.5; Furthermore, the Lys enzyme optimal reaction performed at pH 7.0~8.0 and temperature at 37℃. The recombinant Lys was stable below 40℃ and pH between 3.0 and 6.0. Sr2+ stimulated its activity whereas Ba2+、Ca2+、Zn2+、Cu2+、Mn2+、Mg2+ inhibited the activities. This research accomplished Lys recombinant expression, yield improvement by chemical mutagenesis in K. lactis and characterization of lysostaphin. These research results provide profound guiding significance for the large-scale production and application of recombinant lysostaphin.
In order to prolong the blood half-life of the recombinant ciliary neurotrophic factor in vivo, CNTF was previously formulated by mPEG20k modification and transferrin coupling based on the natural free thiol of Cys17 residue. These two conjugates of PEG20k-CNTF and Tf-PEG5k-CNTF were charicterized and compared using conventional analysis. The HP-SEC and DLS results showed that the conjugates exhibited a similar apparent molecular size. TF-1(CN5α-1) cell survival test showed significant decreased activity of 50.6% (mPEG20k-CNTF) and 65.8% (Tf-PEG5k-CNTF) compared with the unmodified rhCNTF, respectively. Antibody binding affinity revealed that PEG20k-CNTF decreased to 3.8% of the rhCNTF, and Tf-PEG5k-CNTF retained 89.9% of the rhCNTF. Pharmacokinetics study showed that the blood half-lives of PEG20k-CNTF and Tf-PEG5k-CNTF conjugates in vivo were 5.34 ± 0.26 and 8.65 ± 0.60 hours, respectively, which were about 21.4 times and 34.6 times higher than that of rhCNTF. Pharmacodynamics study showed twice weekly administration of 1.0 mg/kg (rhCNTF equivalent) both conjugates led to significant weight-losing, and the efficacy of PEG20k-CNTF group was more potent than that of Tf-PEG5k-CNTF group.
In order to identify the fox, mink, raccoon and dog-derived components in feed, the specific primers and probes for fox, mink, raccoon and dog were designed and synthesized according to the conserved sequence of mitochondrial 16S rDNA. Fluorescent PCR reaction system and reaction conditions were optimized. A number of real-time fluorescent PCR methods were established. Four kinds of animal-derived components were detected in the same PCR reaction system. The developed method could successfully detect four kinds of animal derived materials including fox, mink, raccoon and dog in the same reaction. Through the detection of the source components of 15 other species, the results showed that the primers and probes designed in this experiment had good species specificity. The method has high sensitivity, fox, mink, raccoon and dog DNA detection limit of 0.01ng. Of the 40 samples tested, 5 of them detected raccoon, fox and mink-derived ingredients. The results showed that the method has high specificity and sensitivity. It could effectively identify the fox, mink, raccoon and dog - derived components in the feed, and it was suitable for the related animal products.
Sodium alginate and carboxymethyl cellulose sodium were used as the carriers to immobilize lipase, using ethylene glycol diglycidyl ether (EGDE) and glutaraldehyde as the cross-linking agent, respectively. The results showed that EGDE was superior to glutaraldehyde and was the most effective cross-linking agent. The optimal conditions for lipase immobilized were as follows: sodium alginate 2.5%, carboxymethyl cellulose sodium 1.5%, enzyme loading 800U/ml, CaCl2 5%, EGDE 0.02%, and immobilization time 30 min. Under above optimized conditions, the activity of immobilized lipase was about 380 U/g, and the recovery of the enzyme activity of the immobilized lipase reached about 50.09%. Compared with those from free lipase, the optimal pH (8.0)and temperature (45℃) were higher. The thermal stability of immobilized lipase was better than that of free lipase, and the immobilized lipase could be reused for seven times and still retained about 60% activity.
Graphene (graphene) as a new product in the field of biomedical application is more and more broad, oxidized graphene (graphene oxide, GO) as one of the important derivatives of graphene, due to its different source of electrical structure, can produce fluorescent within a certain wavelength range.This performance is what makes the GO has great potential in biomedical field. The application of the fluorescence properties of GO in molecular detection, disease diagnosis and cell imaging in recent years and prospects of its development were mainly introduced.
Researchs shows that Glypican-3 expresses in hepatocellular carcinoma (HCC) patients with high specificity and is closely related to development of hepatocarcinoma. Including therapeutic antibody,Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) , immunotoxin and vaccine are the focus of targeting Glypican-3 for immunotherapy in HCC at present. The structure and function of Glypican-3 is reviewed, as well as the progress of immunotherapy based on Glypican-3 in HCC is briefly described.
Polysialic acid is a strongly negatively charged, linear homopolymer linked by N-neuraminic acid, present in the human body, animal cells and a few pathogens. Polysialic acid is commonly found in glycoprotein (nerve cell adhesion molecules) and glycolipids. Polysialic acid is a non-glycosaminoglycan, non-immunogenic and biodegradable biomaterial. Polysialic acid can be used as biomaterials in tissue engineering and drug controlled release. Also it can be compounded with other macromolecules to form multi-functional materials. The biological functions, fermentative production and application of polysialic acid are reviewed, in order to provide a hint for the further application of polysialic acid.
Acidophiles can survive in the extremely acidic environment and are most widely distributed in the bacterial and archaeal domains. They play an important role in the bioremediation and source of thermostable and acid-tolerant enzyme. Some neutral engineering strains often encountered the problem of acidification in the process of fermentation. Considering unique acid-tolerant ability of the acidophiles, if the acid-tolerant module from the acidophiles can get and applied to the neutral engineering strains, perhaps the acid-tolerant engineering strains could be built. Therefore, the common mechanisms for acid tolerance in acidophiles, including cell membrane stability and low permeability, energetic metabolism with acid tolerance, repair or protection of macromolecules and cytoplasmic buffering were overviewed, with a view to make some contributions for synthetic biology of acid-tolerant engineering strains.
As the third-generation bioethanol feedstocks, brown algae have received attention because of advantages with high carbohydrate content, short production cycle and haven’t compete with grain for land. However, the improvement of ethanol yield on the basis of low cost is an urgent problem in the actual production of bioethanol. The technical difficulties of large-scale bioethanol production from brown algae were focused on, the research progress of the pretreatment technology and the saccharification and fermentation technology were reviewed, and the prospects of the new potential trend of brown algae bioethanol were also provided.
