DNA methylation and Vitamin C (Vc) play critical roles during the generation of induced pluripotent stem cells (iPSCs). In current study, the interaction between DNMT1 and Vc during reprogramming process was studied. In the absence of Vc, reducing DNMT1 expression with shRNA did not promote the transition from fibroblasts or pre-iPSC to iPSCs obviously. While when Vc was added in the medium, shDNMT1 increased the generation of iPSCs from both kinds of cells. In addition, shDNMT1 actually inhibited cell proliferation and increased the percentage of cells in G1 phase, which reduced its ability to promote reprogramming partially. Therefore, by promoting cell proliferation, decreasing the percentage of cells in G1 phase, and rescuing the inhibitory effect of shDNMT1 on proliferation, Vc further enhanced the ability of shDNMT1 to promote reprogramming.
Objective: To study the reversing effect of Panobinostat on the expression of tumor suppressor gene hepatocyte cell adhesion molecule (hepaCAM) and its synergistic inhibitory effect with hepaCAM on the growth of prostate cancer (PCa) cells. Methods: PC3 cells cultured in vitro were treated with Panobinostat at different concentrations and the effect of Panobinostat on cell proliferation was detected by MTT assay, followed by the expression changes of hepaCAM, histone deacetylase (HDACs), and the acetylation of lys9 of histone H3 (Ac-H3K9) detected by RT-PCR and Western blot. Then after the cells were treated with different factors, cell proliferation activity was detected by MTT assay, cell cycle change was detected by flow cytometry, and gene expressions of cell cycle regulating factors CyclinD1 and proliferating cell nuclear antigen (PCNA) were detected by RT-PCR and Western blot. Results: The inhibitory effect of Panobinostat on the growth of PC3 cells was positively correlated to its concentration and duration. The expression levels of hepaCAM mRNA, its protein, and intranuclear Ac-H3K9 protein were increased, while that of both mRNA and protein of HDAC1, HDAC3, and HDAC4 were decreased when the concentration of Panobinostat increased; Cell growth was significantly inhibited in both the adenovirus-hepaCAM adenovirus alone group and the Panobinostat alone group, with an inhibition rate increased along with the prolonged action time. This inhibition became even particularly noticeable when the two were combined, with a difference of statistical significance (P < 0.05); the percentage of cells in S phase was significantly higher by the combined treatment compared with ad-hepaCAM alone and Panobinostat alone, respectively, with statistically significant differences (P < 0.05). This combination could further downregulate the mRNA and protein expressions of cyclinD1 and PCNA, and the difference was statistically significant (P < 0.05).Conclusion: Panobinostat may enhance the acetylation of lys9 of histone 3 and reverse the hepaCAM expression through its inhibitory effect on HDACs activity in PCa PC3 cells; hepaCAM-expressed adenovirus combined with Panobinostat may synergistically inhibit the growth of PC3 cells, via a potential mechanism associated with the down-regulation of the expression of CyclinD1 and PCNA. This reveals the reasons of hepaCAM deficiency in tumors and provides scientific support for the application of Panobinostat in clinical therapy.
Objective: In order to determine the effects of human truncated variant of hepatocyte growth factor (tvNK1) on carbon tetrachloride (CCl4)-induced liver fibrosis in SD rat. Methods: The recombinant tvNK1 was high-level expression in Escherichia coli BL21 by bio-fermentation and rapid purification of tvNK1 protein using Ni2+-affinity chromatograph. The SD rat model of CCl4-induced hepatic fibrosis was received intraperitoneal injection of tvNK1 for 6 weeks. The mRNA and protein expression levels of Col1A1、Col4A1 and α-SMA in hepatic tissue of different groups were assayed by real-time PCR and Western blot, respectively. The morphology and the collagen fiber of liver sections in different groups were determined by Hematoxylin and eosin (HE) and Masson staining, respectively. Results: The high purity tvNK1 was assayed by SDS-PAGE. The recombinant tvNK1 reduced the mRNA and protein expression levels of Col1A1、Col4A1 and α-SMA in CCl4-induced hepatic tissue in Rat. Moreover, injection with tvNK1 restored the morphology and the collagen fiber in CCl4-induced hepatic tissue in Rat. Conclusion: The recombinant tvNK1 protein attenuated progression of hepatic fibrosis in rat and provided a potential treatment for liver fibrosis and other fibrosis diseases.
Objective: To evaluate the effect of miR-335 target regulating rho associated coiled-coil forming protein kinase 1(ROCK1)on the cell proliferation of ovarian cancer cells SKOV3. Methods: (1)Chose ovarian cancer cells SKOV3 and human normal ovarian epithelial cells IOSE80 as research object. The miR-335 expression was detected by RT-PCR. The ROCK1 protein expression was detected by Western blot.(2)Chose ovarian cancer cells SKOV3 as research object, transfected miR-335 mimic and mimic control respectively. The miR-335 expression was detected by RT-PCR. (3)Chose ovarian cancer cells SKOV3 as research object, the SKOV3 luciferase report carrier and miR-335 mimic were co-transfection to SKOV3, and the targeting effect of miR-335 to SKOV3 was verified by luciferase activity experiment. (4)Chose ovarian cancer cells SKOV3 as research object, which is divided into three groups: SKOV3 group(transfection mimic control), miR-335 mimic group(transfection miR-335 mimic)and miR-335 mimic+ROCK1 group(co-transfection miR-335 mimic+ROCK1). The cell proliferation activity of each group were detected by MTT method. The ROCK1 protein expression was detected by Western blot. The Cyclin D1 expression was detected by RT-PCR. Results: (1)RT-PCR result showed that, the miR-335 expression of ovarian cancer cells SKOV3 significantly lower than human normal ovarian epithelial cells IOSE80(P < 0.05). Western blot result showed that, the ROCK1 protein expression of ovarian cancer cells SKOV3 significantly higher than human normal ovarian epithelial cells IOSE80(P < 0.05). (2)RT-PCR result showed that, transfection miR-335 mimic made miR-335 expression of ovarian cancer cells SKOV3 significantly increased, and significantly higher than transfection mimic control(P < 0.05).(3)Luciferase activity experiment result showed that, transfection miR-335 mimic made the luciferase activity of ROCK1-Wt significantly decreased, but not inhibited the luciferase activity of ROCK1-Mut.(4)After transfection miR-335 mimic, the proliferative activity of ovarian cancer cells SKOV3 and the expression of Cyclin D1 significantly lower than negative control(P < 0.05). After transfection miR-335 mimic+ROCK1, the proliferative activity of ovarian cancer cells SKOV3 and the expression of Cyclin D1 significantly higher than only transfection miR-335 mimic(P < 0.05), but still significantly lower than negative control(P < 0.05). Western blot result showed that, after transfection miR-335 mimic, the ROCK1 protein expression of ovarian cancer cells SKOV3 significantly lower than negative control(P < 0.05). After transfection miR-335 mimic+ROCK1, the ROCK1 protein expression of ovarian cancer cells SKOV3 significantly higher than only transfection miR-335 mimic(P < 0.05), and still significantly higher than negative control(P < 0.05). Conclusion: miR-335 can target ROCK1 to inhibit the proliferation activity of ovarian cancer cells SKOV3.
Objective: To investigate transfect efficiency of CP/DNA at CSC marker CD133+ which is shown to be differently expressed in colon cancer cell lines, and to discuss the relationship between transfect and TR peptide, which is capable to specifically bind to CD133 by targeting CD133+ colon cancer cells. Methods: The expression of CD133+ in four cell lines were divided into 2 groups: the positive colon cell lines in SW480, HCT116, and the negative in CaCO2, SW620. Considering of PEI and Chitosan, they were linked together and composition,particle size,as well as the zeta potential of CP complex was measured.CCK-8 assay was also used to evaluate their toxicity. Using Chitosan-linked-PEI as the vector, PGL3 and EGFP plasmid transfect into four colon cancer cells after 48h incubation, and the CD133-related protein expressions were assessed by Western blotting analysis. Results: The CSC marker expression of CD133+ were significantly different.CD133+ was positive in more than 90% of colon cells, SW480 in 96.97%, HCT116 in 92.96%, however, low expression of CaCO2, SW620 was found in 2.00%, 0.56%.When the N/P of PEI/Chitosan/DNA was 5 ∶4 ∶1, CP complex shows a high transfect efficiency and low toxicity. And when the positive expression of CD133+ was added, the transfect efficiency was decreased. The result of Western blotting analysis showed that the ratios of CD133 in positive group were higher than those in negative group. Conclusion: The transfect efficiency of CP vector can increase the transfect efficient in low expression of CD133+.TR peptide-CD133 may facilitate CP vector into cells.
Steroids from endogenous post-squalene pathway are important precursors for heterologous sterol drugs biosynthesis in yeast. In order to enhance heterologous sterols synthesis, post-squalene pathway should be engineered to increase the compatibility with the heterologous modules. The construction of 7-dehydrocholesterol (7-DHC, the direct precursor of vitamin D3) biosynthesis pathway in Saccharomyces cerevisiae was took as the example to reveal the effect of post-squalene pathway on heterologous sterols production. To produce 7-DHC in Saccharomyces cerevisiae BY4742, ERG6 (encoding sterol C-24 methyltransferase) was knocked out and the Homo sapiens C-24 reductase (DHCR24) together with truncated hydroxymethylglutaryl-CoA reductase (tHMGR) were overexpressed, obtaining strain SyBE0019-Sc-002. Meanwhile, the post-squalene pathway was divided into five modules as ERG1, ERG7, ERG11, ERG24-25-26-27and ERG2-3.Each module was overexpressed in strain SyBE0019-Sc-002 individually. Through GC-TOF/MS analysis and principal components analysis (PCA), it was revealed that overexpression of post-squalene modules brought changes on the contents of the related steroids and effected 7-DHC production. To be specific, overexpression of module ERG11 significantly enhanced the conversion of other steroids to zymosterol; whereas overexpression of module ERG2-3 reduced the accumulation of squalene and significantly increase the synthesis of steroids from lanosterol to 7-DHC. Consequently the highest 7-DHC titer known in shake flask was achieved in the strain with module ERG2-3 overexpressed. Therefore, manipulation of the expression level of module ERG11 and ERG2-3 significantly affected the synthesis of 7-DHC and enhanced the metabolic flux of the whole post-squalene pathway, suggesting ERG11 and ERG2-3 were the potential targets for further optimization 7-DHC producing yeast. This provided a good reference for increasing the compatibility by means of engineering endogenous post-squalene pathway with heterologous sterols synthesis modules.
The SMART algorithing (htpp://smart.emble-heideberg.de) was used for the structural analysis of Streptolysin O (Slo) in Streptococcus pyogenes. The location of protein domain shows that Slo contains a Thiol_cytolysin domain which is composed of 461 amino acids and a transmembrane domain in the amino terminus. A pET101-GENE protein expression system was used to construct genetically engineered bacteria which only express the Thiol_cytolysin domain fusion protein. The recombinant protein was purified using Ni-NTA Resins affinity chromatography. SDS-PAGE analysis of the purified recombinant protein showed that the relative molecular weight of protein is about 50kDa as same as we predicted by bioinformation. Hemolysis testing showed that the purified recombinant protein possessed the hemolytic activity in red blood cells. In addition, the polyclonal antibody against recombinant Slo was prepared in rats. The titre of antiserum was 1 ∶128 000 detected by ELISA. Analysis of Western blot with the polyclonal antibody showed that hemolysin was detected only in culture supermentant of Streptococcus pyogenes. This result showed that recombinant protein is a specific antigen in Streptococcus pyogenes.
Acetoin is the main by-product during the 2,3-butanediol(2,3-BD) fermentation. Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD+. Therefore, intracellular 2,3-BD production is likely governed by the quantities of rate-limiting factor(s) ACR and/or NADH. In bacterial metabolism, glucose is first converted to pyruvate before generation of major products, then pyruvate produced from glucose is channeled into 2,3-BD fermentation pathway. While, glucose can be converted to pyruvate through two main metabolism pathway PPP and EMP. However, there are NADPH generated via the former pathway, which was not necessary for 2,3-BD production. Glucose-6-phosphate dehydrogenase(G6PDH) is the key enzyme in PPP pathway and to enhance the level of intracellular NADH, metabolic flux of EMP pathway was increased by disrupting G6PDH, an key enzyme in PPP pathway. Flask fermentation experiments indicates that the level of intracellular NADH was enhanced in the recombinant strain B. subtilis168△zwf, and the yield of 2,3-BD was increased by 15.0%, and the titer of main by-products AC was decreased by 10.6%, however, the accumulation of acetic acid, lactic acid and other organic acid were enhanced. In order to improve the yield and production efficiency of 2,3-BD further, a recombinant strain B.subtilis168△zwf/pMA5-kphs was constructed by overexpressing ACR in B.subtilis168△zwf. Compared with parental strain, the yield of 2,3-BD was increased by 37.3 %, AC was decreased by 28.1%, and acetate acid, lactate acid, succinic acid and other by-products also were decreased.
With the advances in cell culture expression levels and production capacity in the last two decades,it is crucial to improve the binding capacity of protein A resin and decrease processing time. Then the dual flowrate loading strategy is proposed. A cubic equation of GE MabSelect dynamic binding capacity(DBC) was created by measuring several DBC values of the retention time with WLB303 mAb. A unique DBC table of GE MabSelect was generated subsequently to design dual flowrate loading strategy. The fastest dual flowrate loading mode was firstly confirmed by using purified WLB303 mAb to practice on a 2.7 ml column. And it was demonstrated to show obvious promotion in process efficiency by using clarified harvest to practice on a 7 ml column.
A rapid and simple analysis method based on uv-vis spectrophotometry technology was established to measure the concentration of free sulfhydryl groups in recombinant basic fibroblast growth factor. Detection reagents,standard references and samples were mixed, incubated for some time,followed by OD412 value reading with uv-vis spectrometer. Standard curve was generated by taking the concentration of standard reference as the horizontal coordinate, taking the OD412 values as the vertical coordinate. The concentration of free sulfhydryl groups in tested samples was calculated according to standard curve. The method qualification results showed that the method is free sulfhydryl specific, with accuracy range 91.7%~107.4%, precision range 5%~8%, and linearity range 20~80μg/ml. The measured result of free sulfhydryl groups on molecular surface or total were consistent with the theoretical value.This method can be used to measure the free sulfhydryl groups in proteins to monitor the correctness and homogeneity of molecular structure during the quality research of related biopharmaceuticals development.
Rhodotorula mucilaginosa is an oleaginous ocean yeast that of great interest for both lipid and carotenoids production. However, rationally engineering of this cell factory is impeded due to the absence of efficient and reliable genetic tools. Agrobacterium-mediated transformation (ATMT) was successfully developed for R. mucilaginosa. First, the promoter of glyceraldehyde-3-phosphate dehydrogenase (GPD) was identified by referring to the completely annotated genome information of Rhodosporidium toruloides. Then, the codon optimized hygromycin (hyg) gene under GPD was used as both selection marker and functional gene through ATMT transformation in R. mucilaginosa, the integration was confirmed by phenotype, genotype and Western blot analysis in protein level. The results provided a practical method for functional integration and expression of hyg genes in R. mucilaginosa, which would facilitate the development of genetic tools.
Induced pluripotent stem cells (iPSCs) are a type of cells similar to embryonic stem cells (ESCs) which have the characters of self-renewal and pluripotency.They can be obtained through the induced reprogramming of differentiated somatic cells.iPSC has broad application prospects in the establishment of disease models in vitro,regenerative medicine,embryology etc. The controversies and obstacles that challenge the field are its tumorigenicity,low transformation efficiency and the low degree of fitting of the disease models. The majorizations of method on reprogramming were summarized, and the ways to increase transformation efficiency and reduce tumorigenicity were emphasized.The application prospects of iPS in clinical therapy and scientific research in the future were explored
With the development of tissue engineering, the use of mesenchymal stems cells (MSCs) differentiation into chondrocytes, to treat the articular cartilage injury, induced by osteoarthritis or joint trauma, has been attracting more and more attention. Hyaluronic acid (HA), a acidic polysaccharide macromolecules, is one of the main components of cartilage matrix and possess some characteristics such as biocompatibility and biodegradability, and can be used as excellent natural biological material. It has been a long history of HA which is used as scaffold material on repair cartilage defect. In recent years, some studies found that HA not only could be used as scaffold material, but also a regulator factor, applied to differentiate into cartilage. The application of MSCs differentiating into cartilage cells combined with HA in recent years were outlined. It will provide new idea for the clinical application of tissue-engineered cartilage on the basis of the MSCs.
The cellular membrane as a selectively permeable barrier plays an important role to maintain a relatively stable intracellular environment. But this limits the entry of biological macromolecules and drug into cells and have a obstacle in diagnosis and treatment of some of intracellular disease and drug-target applications. How to carry biological macromolecules and drugs through the cell membrane into the interior of the cell has also been a hot and difficult medical research. Cell-penetrating peptides(CPPS) are a class of diverse peptides and can carry polypeptides, proteins, nucleic acids, nanoparticles, virus particles and drugs through the cell membrane into cells, resulting in a complete cargo internalization. CPPS as a carrier with a high transduction efficiency and low toxicity characteristics have gotten widespread concern and lots of researches. These peptides as delivery vectors have a potential diagnostic and therapeutic applications in fluorescence imaging, cancer therapy, anti-inflammatory therapy and targeted therapy drugs.
γ-poly-glutamic acid (γ-PGA) is a natural,biodegradable,new anionic polymer and synthesized by some bacillus and archaea and one eukaryote.They are divided into glutamate-dependent and non-dependent glutamate.The gene required for γ-PGA contain capsule and polyglutamate synthase.The synthesize organized by the responding expressed protein regulates the synthesis and transport of γ-PGA.γ-PGA is friendly to environment with characteristics of good water solubility, moisture resistance, water absorption, good biological compatibility and biodegradable, edible and environmental pollution, etc..Therefore,γ-PGA and its derivatives have been of interest in a broad range of application prospects such as medicine, agriculture, food, environment, cosmetics. The structural characteristics of γ-PGA, the microbial synthesis related genes, synthesis mechanism, application, mutagenic treatment were focused on.Using technology of physics and chemistry to mutagenize can achieve high-yielding strains of γ-PGA,which provide the basis for improving production of γ-PGA.
Being antibacterial chemicals, antibiotics play a very important role in modern medication. The Chinese pharmacopoeia was revised in 2015 which included 363 antibiotics, and introduced four methods to measure the content of antibiotics. A summary of antibiotics measurement methods showed that among the 363 antibiotics, 275 antibiotics are measured using high performance liquid chromatography, 75 antibiotics are measured using microbial assays, 8 antibiotics are measured using ultraviolet-visible spectrophotometry, and only 5 antibiotics are measured by titration. A summary of methods for the quantification of the content of antibiotics based on the latest Chinese pharmacopoeia revised in 2015 was provided and analyzed, hoping to provide a reference in the determination of the content of residual environmental antibiotic.
