Objective: To explore whether Calcyclin S100A6 in tumor microenvironment promotes cell proliferation of colorectal cancer(CRC) through affecting macrophages (Mφ) and its mechanism. Methods: Prokaryotic expression was used to prepare recombinant human protein GST-S100A6 (rS100A6) and GST (as control). THP-1 were induced to Mφ by PMA (Phorbol-12-myristate-13-acetate). A6-Mφ were the macrophages which were treated with rS100A6 for 24h. The proliferation of CRC HCT116 cells was detected by Trypan blue staining, CCK8 and crystal violet staining. IL-6 mRNA and protein level in A6-Mφ were tested with quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The protein levels of total JAK2 and STAT3 (t-JAK2 and t-STAT3) and the phosphorylated JAK2 and STAT3 (p-JAK2 and p-STAT3) in HCT116 cells were detected by Western blot. Results: (1) rS100A6 and GST were prepared successfully. (2) A6-Mφ promoted proliferation of HCT116 cells (P<0.05). (3) rS100A6 upregulated IL-6 expression in macrophages (P<0.05). (4) IL-6R blocking antibody partly reversed the facilitation of A6-Mφ to proliferation of HCT116 cells (P<0.05). (5) A6-Mφ increased protein levels of p-JAK2 and p-STAT3 in HCT116 cells (P<0.05), but not t-JAK2 and t-STAT3. Conclusion: S100A6 in tumor microenvironment facilitates proliferation of HCT116 cells through upregulating IL-6 expression in macrophages and activating IL-6/JAK2/STAT3 pathway in HCT116 cells.
Objective: miR-219 mediated the development of acute myocardial infarction through the regulation mechanism of TGFBR2 and the ENDMT pathway. Methods: The expression of miR-219 in the serum of AMI patients and mice was detected by qRT-PCR. The target gene of miR-219 was screened in gene database of microRNA. The regulatory mechanism of TGFBR2 and miR-219 was examined by luciferase reporter gene and qRT-PCR.The blood scores (LVEF) of AMI mice were measured 4 weeks after myocardial injection of miR-219 lentivirus (LVEF) by cardiac echocardiography. The mRNA expression of Nppa in AMI mice injected with miR-219 lentivirus was detected by qRT-PCR. The changes of left ventricular fibrosis in mice were detected by Masson’s trichrome staining. immunohistochemical analysis of the left ventricular section of mice was performed using spla-sma. Expression levels of P-smad2, P-smad3 and TGFBR2 hypoxia induced protein phosphorylation were detected by Western blot. Results: miR-219 can regulate AMI. miR-219 inhibited the mRNA expression of TGFBR2, while miR-219 inhibitor inhibited the down-regulation effect. miR-219 can inhibit the process of acute myocardial infarction and promote the recovery of myocardial function of infarction. miR-219 can promote the angiogenesis and maturity of the myocardial tissue of AMI mice, and eventually the cardiac contractility increases and the cardiac function recovers. miR-219 can inhibit the inhibition of EndMT pathway by TGFBR2, leading to the alleviation of the pathological process of AMI. Conclusion: miR-219 can inhibit the EndMT pathway by inhibiting TGFBR2, reduce myocardial fibrosis, promote angiogenesis and maturity, recover cardiac function and inhibit the pathological development of AMI.
The influence of delayed activation on in vitro and in vivo porcine somatic cell clone was studied in order to enhance the efficiency of porcine clone so as to obtain more clone pigs. Related studies had shown that, compared with synchronous fusion activation, delayed activation could significantly improve the cleavage rate (P<0.01) and the blastocyst rate (P<0.05) of cloned embryos, although it might decrease the fusion rate of cloned reconstructed embryos (P> 0.05). CB was utilized to assist in the delayed activation for 4h to reconstruct embryos. The blastocyst rate of the delayed activation group was significantly higher than that of the group not using CB (P<0.01). Cloned embryos were transplanted into 126 recipient sows. It was shown that the parturition rate of recipient sows was remarkably higher in the delayed activation group than in the synchronous activation group (P<0.05). Although these two groups did not differ significantly in terms of average litter size, average live births and clone efficiency, it was obvious that more cloned piglets were obtained in the delayed activation group than in the synchronous group. The above-mentioned result demonstrates that delayed activation may enhance the in vitro and in vivo development efficiency of porcine clone embryos.
Objective: To optimize the prokaryotic expression conditions of AMPs17 recombinant protein and analyze the antifungal activity of recombinant protein. Methods: Compare different induction temperatures (25℃, 28℃, 30℃, 32℃, 34℃), isopropylthio-β-D galactoside (IPTG) induced concentration (0.025mmol/L, 0.05mmol/L, 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L) and induction time (12h, 15h, 18h, 21h, 24h) on the expression of AMPs17 recombinant protein, screening the optimal expression conditions of AMPs17 recombinant protein. The recombinant protein was purified by nickel ion metal chelator affinity chromatography column, and the expression results were analyzed by SDS-PAGE electrophoresis and ImageJ image analysis system. The recombinant protein of AMPs17 was identified by Western blot and the purity of the recombinant protein was analyzed by high performance liquid chromatography(HPLC). The antifungal activity was detected by a micro liquid dilution method and a colony counting method. Results: The results showed that the expression of AMPs17 recombinant protein was the highest and the most stable when induced at 32℃ and IPTG concentration of 0.05mmol/L for 15h. The HPLC analysis showed that the purity of AMPs17 recombinant protein reached 90%. In addition, AMPs17 recombinant protein can effectively inhibit the growth of Candida albicans. Conclusion: The induction and expression conditions of antibacterial peptide AMPs17 were optimized, and proteins with high expression, stability and antifungal activity were obtained, which provided certain experimental basis for the follow-up antibacterial mechanism and application research.
Human mitochondrial O-GlcNAc transferase (mOGT) localized in mitochondria inner membrane through its N-terminal martrix targeting sequence(MTS), mOGT overexpression caused cell spoptosis. O-GlcNAc modification existed in most eukaryote cells except Saccharomyces cerevisiae. In order to analyze mOGT induced mammalian cell apoptosis, the mechanism of mOGT caused yeast cell growth defect was investigated. Herein, mOGT was overexpressed in Saccharomyces cerevisiae. Both yeast growth defect and mOGT localization were largely depends on MTS sequence. Furthermore, MTS expression caused yeast mitochondria fussion. Therefore, MTS overexpressed yeast cells might be applied to analyze mOGT caused cell apoptosis.
Macroporous absorbent resin DA-201 was adopted as a carrier to immobilize marine-derived lipase, and the influence of additives on the immobilization process was explored. NH4Cl, mannose and glycine were used as the additives, and the conditions were optimized by a combination of single factor and orthogonal experiments. The results showed that the optimal conditions with additive NH4Cl were: citric acid-sodium citrate buffer pH6.0, immobilization temperature 30℃, carrier quantity 0.5g, NH4Cl concentration 25mmol/L, immobilized time 3.0h; enzyme activity reached 115.27U/g, which was 47.42% higher than that of the immobilized enzyme without additive. The optimal conditions with additive mannose were: potassium dihydrogen phosphate-sodium hydroxide buffer pH7.0, immobilized temperature 35℃, carrier quantity 0.5g, mannose concentration 10mmol/L, the immobilized time 4.5h; the enzyme activity reached 122.75U/g, which was 6.50% higher than that of the immobilized enzyme without additive. The optimal conditions with glycine additive: potassium dihydrogen phosphate-sodium hydroxide buffer pH7.0, immobilized temperature 20℃, carrier quantity 0.5g, the glycine concentration 25mmol/L, the immobilized time 7.5h; the enzyme activity reached 141.69U/g, which was 26.12% higher than that of the immobilized enzyme without additive. The addition of different additives exhibited great effects on the immobilization through absorbtion by macroporous absorbent resin DA-201 and could greatly improve the adsorption efficiency. Additionally, buffer type, pH, temperature, additive concentration and immobilization time were found to have great influence on the adsorption of lipase by resin DA-201, which provides good reference for subsequent research of the immobilization of industrial enzymes.
The expression cell lines constructed by random integrating target gene into mammal cell’s genome may not express the target gene stably over passages because the target gene might be inserted into unstable region of chromatin, which is known as position effect. To solve this problem, site specific integration of target gene (Human serum albumin gene) into stable hot spot of CHO chromatin by using homologous dependent recombination (HDR) method mediated by CRISPR/Cas9 can be effective, because the position effect issue can be overcome. Here, two site specific integration hits of human serum albumin gene were obtained verified by conducting 5' junction PCR, 3' junction PCR and out-out PCR. The Western blot results revealed target protein could be detected in the supernatants of culture; the average amount of HSA protein expressed per cell per day was around 0.5pg cell/d over different cell passages (passage 3, 12, 23, 35, 50) at adherent cell mode for both two hits. One hit was adapted to suspension culture. The expression level of this hit at batch mode in different cell passages (passage 1, 25, 50) were stably around 13-14mg/L.It was feasible to insert heterogenous gene into the stable hot spot of CHO cell line and corresponding gene expression level was stable over passages.
Objective: The whole-cell biocatalyst, overexpressing diacetyl reductase (DAR) and introduced in situ-NADH regeneration systems was applied to improve (S)-acetoin production from prochiral diacetyl.Methods: The gene encoding DAR from Paenibacillus polymyxa was cloned and expressed in Escherichia coli. Recombine DAR was purified by HiTrap TALON affinity chromatography, then enzyme activities and molecular kinetic parameters of purified DAR were measured. NADH in situ regeneration system based on glucose dehydrogenase (GDH) from Bacillus subtilis was introduced. The whole-cell biocatalyst, overexpressing DAR and GDH was applied to (S)-acetoin produce and the reaction conditions were optimized.Results: DAR showed a high catalytic efficiency and enantioselective (enantiomeric purity 95.86%). The Km, Vmax and Kcat values of DAR for diacetyl were 2.59mmol/L, 1.64μmol/(L·min·mg) and 12.3/s, respectively. The whole-cell biocatalyst, introduced in situ-NADH regeneration systems resulted in higher (S)-acetoin concentration, productivity and yield form diacetyl. Under optimal conditions in fed-batch bioconversion, 51.26g/L (S)-acetoin was produced from 63g/L diacetyl with a productivity of 5.13g/(L·h).Conclusion: The compound of prochiral diacetyl was used as substrate for asymmetric synthesis of high value chiral (S)-acetoin. The results demonstrated that whole-cell biocatalyst, introduced in situ-NADH regeneration systems, can effectively improve the production of (S)-acetoin with good applicability and economic performance.
Objective: Extracellular production of recombinant proteins in Escherichia coli is limited by the inefficiency of inherent secretion system. An inducible cell lysis system was designed and constructed to enhance secretion of intracellular recombinant protein in E.coli.Methods: Considering colicin E7 could promote cell lysis, E.coli cell lysis systems were constructed by co-expressing target protein and colicin E7 lysis to release the recombinant proteins to culture medium.Results: A reporter protein (red fluorescent protein, RFP), as a recombinant protein, was co-expressed with E7 in E.coli to evaluate the cell lysis systems. Expression of recombinant protein was controlled by T7 promoter. While E7 cassette was controlled by two promoters (T7 promoter and araBAD promoter) which determined the expression timing of E7 and therefore determined the timing of cell lysis. Compared to one-step induction by IPTG, the two-step induction by IPTG and L-arabinose was better for the production and secretion of recombinant proteins. The two-step inducible lysis system was also used to express zearalenone (ZEN) degrading enzyme, and high enzyme activity was detectable in the culture supernatant samples. The secreted enzyme could degrade about 5.8μg ZEN in 30minutes at 37℃.Conclusion: The colicin E7 assisted two-step inducible cell lysis system could be potential for expression of recombinant proteins and their secretion to extracellular in E. coli.
Mycoplasmal pneumonia of swine (MPS) is a severe respiratory disease of pig which is caused by Mycoplasma hyopneumoniae (Mhp) worldwide. The disease decreased feed conversion efficiency causing significant economic loss. Accurate, sensitive and quick detection method is much helpful for understanding the prevalence of Mhp in pig farms, and also can improve the preventive and therapeutic measures, and management practice. Etiological, molecular biology, and serological detection methods of Mhp were reviewed. Comprehensive data of Mhp detection methods for scientists was provided.
PRDM16(PR domain-containing 16) is a 16th member of PR domain family, was firstly found in a patient with leukemia and was initially thought to be related to myelodysplastic syndrome(MDS) and chronic myelogenous leukemia(CML). PRDM16 contains six important functional domains, including PR domain(PR), zinc finger domain 1(ZF-1), proximal regulatory region(PRR), repression domain(RD), zinc finger domain 2(ZF-2), and acidic activation domain(AD), respectively. The ablation of PR domain which is an exclusive domain for PRDM family has been linked to MDS and CML; the domains of ZF-1 and ZF-2 are capable to bind to peroxisome proliferator-activated receptor-α/γ(PPAR-α/γ), CCAAT-enhancer binding proteins-β(CEBP-β), peroxisome proliferator-activated receptor-γ coactivator1-α/β(PGC1-α/β) and mediator complex subunit 1(MED1); the RD domain is main site for PRDM16 to bind with C-terminal binding protein-1/2(CtBP-1/2). In mammals, the PRDM16 is involved in a spectrum of biological processes including cell fate determination and development. The PRDM16 is capable to regulate transcription via intrinsic chromatin-modifying, complexing with histone-modifying. Studies have shown the pivotal roles of PRDM16 in the determination and functions of brown and beige fat cell, as well as in thermogenesis, hematopoiesis and cardiac development. Studies indicated that PRDM16 is a key transcriptional regulator in differentiation of the brown adipocytes. PRDM16 has been found has an important role in maintaining “brown fat cell-specific” morphological properties and biological functions, as well as in association with high abundant mitochondria contents and its thermogenesis capacity. PRDM16 controls a “bidirectional cell fate switch” for skeletal myoblasts and brown fat cells. It involves in a “browning” process in white adipose tissues, which transform white adipocytes into brown/beige adipocytes. PRDM16 has also been related to the increase of visceral fat which may cause the immune response in animals. In the process of “browning”, many transcriptional factors were recruited to the promoter or enhancer regions of brown fat-related genes by regulation of PRDM16 through its ZF-1/2 domain. This, in turn, eventually promotes genes expression and BAT differentiation. And the recruit of C-terminal banding protein in the promoter of white fat cell-related genes makes them repressed by PRDM16 via its PLDLS motif in the repression domain. Recently, the genetic variations in the PRDM16 gene were identified, in humans and livestock and have been associated with a spectrum of diseases and production traits. Those reported SNPs in PRDM16 were summarized. The SNPs in human PRDM16 were significantly associated with risk of diseases such as dilated cardiomyopathy, dyslipidemia, migraine without aura and metabolic syndrome. In livestock, variations of PRDM16 have been mainly associated with growth traits and other important economic traits, including body weight, body size and carcass weight. Because of the potential applications of PRDM16 in treating human diseases and improving economic traits in livestock, the future research areas may focus on understanding the mechanism underlying the action of PRDM16 in adipose biology which may have relevance to other PRDM family members. This new knowledge may also have the potential to be exploited for therapeutic and breeding benefits.
Induced pluripotent stem cells(iPSCs) refer to stem cells that are artificially produced by cellular reprogramming, which have similar functions to embryonic stem cells. They can differentiate into all cell types, and avoid the ethical controversy of ESCs and immune rejection after transplantation. They have a broad application prospect.The advances in the in vitro differentiation of male germ cells using iPSCs by different inducers was reviewed, the effect was also investigated. Exploring development mechanisms of germ cells is promising to promote future reproductive and developmental engineering technologies.
The monoclonality of recombinant cell lines is one of the important factors to ensure the quality of the product, so it has received more and more attention. Currently, some drug regulatory agencies require the application organization to demonstrate the monoclonality of the cells via appropriate experimental. The process and common methods (such as limiting dilution cloning, ClonePix, and fluorescent-activated cell sorting) for the clone selection of engineering cell lines for production were introduced.How to ensure the monoclonal of engineering cell, and the significance of engineering monoclonal in the production of the biological drug were discussed.
L/D amino acids are common components in bacteria. Unlike L-amino acids (L-AAs), D-amino acids (D-AAs) are not involved in protein synthesis, but participate in the biosynthesis of cell wall peptidoglycan, directly affecting the shape, quantity and strength of bacterial cell wall. D-AAs play an important role in bacterial characterization, drug bacteriostasis, and target determination. At present, the mechanism that exogenous addition of D-AAs is involved in peptidoglycan synthesis has been studied and fluorescent derivatives applied to bacterial visualization, specifically detecting cell wall formation/remodeling, bacterial growth and cell morphology. However, the mechanism of how D-AAs affect bacterial growth and its resistance to stress has not been elucidated. The current studies of D-AAs was summarized. The biosynthesis pathway of D-AAs in bacteria, mechanism of cell wall synthesis and the effects of non-canonical D-amino acids on bacterial regulation, and its application in bacterial visualization were highlighted. In the end, the future research direction of D-AAs are prospected.
Synthetic biology, which integrates such disciplines as biology, engineering, chemistry and information technology, is an emerging field and considered as a disruptive technology in the field of biotechnology. It enables the modification or even creation of biological organisms, and has great potentials to be widely applied in the fields of medicine, pharmacy, agriculture, material science, and energy. Related development strategies, funding projects and policies of synthetic biology in major countries and regions were analyzed; the development trends of patented technology, reveals the distribution of research hotspots, and conducts a comparative analysis on the numbers and influences of patents in major countries and institutions were summarized. The results can provide references and implications for scientific researchers and decision makers in the field of synthetic biology in China.
