Objective: To construct a CHD5 gene overexpressing lentiviral vector using CRISPR/Cas9-SAM system and analyze its effect on the proliferation, migration and invasion in bladder cancer cell line T24.Methods: Three pairs of sgRNAs (sgRNA1, sgRNA-2, sgRNA-3) were designed targeting CHD5 gene. The sgRNA was inserted into the LV-sgRNA-MS2-P65-HSF1-Neo vector, and the lentivirus particles with high titer were obtained after 293T cell packaging. Subsequently, bladder cancer cell line T24 were infected with lentivirus at MOI=10. The expression of CHD5 mRNA and protein in T24 cells was examined by RT-qPCR and Western blot, respectively. CCK8 assay, flow cytometry, wound healing and transwell assay were performed to detect the effects of overexpression of CHD5 on the proliferation, apoptosis, migration and invasion of T24 cells.Results: The CHD5 overexpressing lentiviral vector was successfully constructed. The results of RT-qPCR and Western blot confirmed that the mRNA and protein expression of CHD5 in T24 cells were significantly higher than those in the blank and negative control groups after lentivirus infection(P<0.05), and the effect of sgRNA-3-MS2-P65-HSF1 was the most significant.CCK8 and flow cytometry analysis showed that overexpression of CHD5 inhibited the proliferation and promoted apoptosis of T24 cells, which had statistical significance compared with the control group (P<0.001). Wound healing and transwell assay showed that overexpression of CHD5 inhibited the migration and invasion of T24 cells (P<0.01).Conclusion: CHD5-SAM lentivirus has been successfully constructed. Overexpression of CHD5 could induce apoptosis, inhibit proliferation, migration and invasion of bladder cancer cell line T24.
To establish and identify the neocortex and hippocampus specific GABAA receptor γ2 subunit (GABRG2) knockout mice via Cre/Loxp conditional gene knockout technology, and provide the important animal model for further investigating the functional role of GABRG2 in hippocampus and neocortex GABRG2 in epileptogenesis. The constructed GABRG2 fl/wt mice and the mice that express Cre recombinase to approximately 88% of the neurons of the neocortex and hippocampus were bred and authenticated, respectively. Mating and identification of GABRG2 fl/fl mice with Cre mice were carried out, and the GABRG2 fl/wt Cre + mice were screened. The mouse genotypes were identified by PCR. The Real-Time PCR and Western blot were used to detect the expression of GABRG2 mRNA and protein levels in mouse neocortex and hippocampus. PCR results indicate that mouse genotypes are consistent with GABRG2 fl/wt Cre +. Compared with control group mice, GABRG2 mRNA and protein levels were significantly reduced. During the temperature elevation, the GABRG2 fl/wt Cre + mice had significant seizures. Based on the Cre/loxp conditional gene knockout technology, that succeed in building a group of neocortex and hippocampus specific GABRG2 gene knockout mice which can stably go down to the future generation, which would supply a technical basis for animal models in further researches of the regulation and mechanism of GABRG2 gene in the progress of epilepsy.
Objective: To investigate the effect of miR-186-5p on the proliferation and differentiation of mouse 3t3-L1 preadipocytes and its potential molecular mechanism.Methods: qRT-PCR was utilized to measure the expression levels of miR-186-5p in adipose tissue of different-age mice or during the proliferation and differentiation of 3T3-L1 preadipocyte; miR-186-5p mimics or inhibitors were transfected into the 3T3-L1 adipocytes cultured in growth or differentiation medium using lipofectamine; CCK-8,EdU, qRT-PCR or Oil red O staining were performed to determine the effect of miR-186-5p on 3T3-L1 preadipocyte proliferation or differentiation, respectively. Meanwhile, TargetScan and dual-luciferase assay were used to predict and confirm the target genes of miR-186-5p.Results: With aging of mice or 3T3-L1 preadipocyts differentiation into mature adipocyte, the mRNA levels of miR-186-5p were upregulated gradually. When compared to the negative control, notably, mimics or inhibitors transfection could remarkably increase or decrease the expression levels of miR-186-5p, respectively. MiR-186-5p overexpression significantly inhibited the 3T3-L1 preadipocyte proliferation and promoted its differentiation, and in contrast, miR-186-5p inhibition had opposite effect on 3T3-L1 preadipocyte proliferation and differentiation compared to the overexpression group. Further,overexpression of miR-186-5p significantly repressed the relative luciferase activity of wild type-Wnt5a and Mapk1 3'-UTR were found, whereas its inhibition effect was abolished by the mutation of the binding sites.Conclusions: miR-186-5p inhibits the proliferation of 3T3-L1 preadipocyte and promotes its differentiation by targeting Wnt5a and Mapk1.
Objectives: To express human Nek2 protein by prokaryotic expression system, optimize expression conditions and purify Nek2 protein and prepare anti-Nek2 polyclonal antibody.Methods: The fragment of Nek2 gene was constructed on the prokaryotic expression vector pET30a(+) and transformed into E. coli BL21 (DE3).Expression of the recombinant protein was induced with IPTG and the conditions such as induction temperature, IPTG inducer concentration and induction time were optimized.The protein was purified by staining with 250mmol/L KCl after 12% SDS-PAGE, and the purified Nek2 protein was identified by mass spectrometry.Polyclonal antibody was prepared by immunizing BALB/c mice with purified Nek2 protein, and the titer and specificity of polyclonal antibody was detected by ELISA, Western blot and immunofluorescence assays.Results: The recombinant prokaryotic expression plasmid pET30a(+)-Nek2 was constructed and the recombinant human Nek2 protein was mainly expressed in the form of inclusion bodies.Optimal induction conditions were 28℃,180r/min,IPTG concentration 0.2mmol/L and 32h induction for expression of recombinant protein.The purified protein by mass spectrometry was Nek2 protein, and the concentration of the purified Nek2 protein was 1.35mg/ml.The purified Nek2 protein was used to immunize mice. The titer of polyclonal antibody was greater than 1:243 000 and the polyclonal antibody exhibited a perfect antigenic specificity.Immunofluorescence assay showed that Nek2 was mainly localized in the cytoplasm and nucleus.Conclusion: The recombinant Nek2 protein was used to obtain a anti-Nek2 polyclonal antibody with perfect antigenic specificity.
Canine brucellosis is a zoonotic disease that causes livestock abortion and human Malta fever worldwide. Recently, with the increase in the number of related host animals such as dogs, infection reports of family children are not uncommon, so study Brucella canis has important public health implications. Using 91 strains around the world, Bayesian method was used to cluster the strains, and based on the core-SNPs and molecular clock model, the evolutionary tree was constructed for population structure and spatiotemporal distribution analysis, and COG function clustering was used to study the functional differences. The four phylogenetic groups (PG) found were significantly associated with their geographic origin. Among PG1-3, there was transmission from Asia to Africa, Europe and America. PG4 was colonized in North America, and its drug resistance genes were gradually missing. Functional gene has different presence/absence profiles in 4 PGs, PG3 is the most complete, and other PGs have unique functional gene absence, especially PG2 mostly lacks ABC-type transport system components. Understanding the global distribution and spread of Brucella canis and its genomic variation will help develop new diagnostic and vaccine targets to combat the epidemic caused by Brucella canis.
Fusarium oxysporum f.sp.Lini, a forma specialis of Fusarium oxysporum on flax, is soil-borne fungus which causes Fusarium wilt of flax, a serious disease of flax resulting in great loss of yield and quality of the crop. It has been identified that C2H2 zinc finger transcription factor Pcs1 in Fusarium graminearum is responsible for the generation of conidia produced from intercalary phialides on hyphae. Objective:The gene disruption of the Folpcs1 is performed according the principle of homologous recombination for identifying the function of the Folpcs1, the homologous gene of the pcs1, in Fusarium oxysporum f. sp. Lini. Methods:The disruption of the gene was made by the Split-Marker strategy after the genomic and complementary DNA was sequenced. The deletion cassettes containing a hygromycin resistance gene (hph) was constructed and transformed into protoplast of the wild type of the fungus mediated by polyethylene glycol. The deletion mutants, ΔFolpcs1s, were obtained and confirmed by PCR using positive and negative primer. To make complementation for the deletion mutuant, the coding frame sequence of the Folpcs1 gene, together with its upstream and downstream flanking sequence, was cloned into the pZWH1 containing neomycin resistant gene and transformed into the deletion mutant. Results:The sequencing revealed that the gene contained one intron of 654bp and the whole cDNA sequence consisted of 2 846bp. The morphological and microscopic observation found that growth rate of deletion mutant significantly reduced and the conidium was hardly observed in culture mixture of the mutant. The revertant recovered in normal growth and conidiogenesis of the wild type. Conclusion:The results suggest that Folpcs1 is responsible for asexual reproduction and vegetative growth in Fusarium oxysporum f. sp. Lini.
The residues of natural polysaccharide substrates were often substituted by acetyl groups, and acetyl esterase can cut acetyl groups on these substrates, which is conducive to the further degradation. The gene aesA which encoding an acetyl esterase was cloned from mannan utilization gene cluster of Bacillus sp. N16-5 and heterologously expressed in prokaryotic host. The gene aesA is 957bp long and encodes 318 amino acids, belonging to the carbohydrate esterase family 7 (CE7). AesA showed good catalytic activity for 4-methylumbelliferyl-acetate and pNP-acetate, however, there was no active effect on alpha-naphthyl acetate. The enzyme activity for 4-methylumbelliferyl-acetate is 1.68U/mg, and the kinetic parameters Km, Vmax and kcat/Km, were measured by 3.27mmol/L, 0.044mmol/min and 289.71ms-1, respectively. Metal ions Fe3+, Fe2+, Mn2+ and Cu2+ all promoted the activity of AesA, and Cu2+ exhibited the most significant promoting effect. AesA has a significant synergistic effect with β-mannanase ManA on degrading acetylated mannan substrates, and the synergy degree reached 1.47 when using acetylated locust bean gum as substrate. It is helpful to understand the mannan utilization mechanism of Bacillus sp. N16-5, and has potential application prospects in mannan degradation.
Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the synthesis of acetyl-CoA from pyruvate and coenzyme A (CoA) using thiamine pyrophosphate (TPP) as coenzyme. The four subunit-type TnPFOR from T. neapolitana was expressed in Escherichia coli and characterized. The gene of TnPFOR from T. neapolitana was cloned into pET-20b(+). TnPFOR was purified by a heat treatment followed by an ion exchange chromatography. The TnPFOR had an optimal condition for its maximum activity at 90℃ and pH 6.5 and it was indeed thermostable with a half-life of more than 1h at 90℃. The application of TnPFOR to catalyze the conversion of pyruvate into acetyl-CoA was also evaluated. The influence of different reaction conditions (reaction temperature, pyruvate concentrations and reaction time) on the synthesis of acetyl-CoA was discussed. The optimal reaction temperature is 90℃, pyruvate concentrations is 1.5mmol/L and the reaction time is 2min.
The trehalose synthase (EC 2.4.1.245) from Myxococcus sp.V11 (TreS II) catalyzes the reversible interconversion of maltose and trehalose. The high catalytic activity and high conversion rate of maltose into trehalose of TreS II indicate that it has potential application in industrial production of trehalose. However, the thermal instability of TreS II limits its wide application in trehalose production. Objective:The effects of amino acid residues mutations on the thermal stability, optima of pH and temperature, and specific activity of TreS II were studied by site-directed mutagenesis. Methods: Site-directed mutation experiment of the two possible metal ion-binding sites (A283 and Y537) and the three sites (Q3, W374 and R449) in two regions which may correlate with thermostability by using overlapping PCR were performed. Mutants of A283R, Y537H, Q3D, W374D and R449Q were heterogeneous expressed in E.coli BL21(DE3). At the same time the specific activity, the optimum reaction temperature, the optimum pH and the thermal stability of mutants were compared with wild-type strain. Results: Mutation of Q3D, W374D, R449Q, A283R and Y537H enhanced the thermal stability, but did not affect the pH and temperature optima. Only the mutant R449Q reduced the specific activity. The modified enzymes A283R and Y537H showed 68% and the mutants Q3D, R449Q, W374D showed 35% of maximal activity after incubating in maltose substrate for 3h at 60℃ compared to only 20% activity for wild-type enzyme. Conclusion: These factors may render TreS II relatively more thermostable among mesophilic trehalose synthases. The thermophilic amino acid residues provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.
Epoxide hydrolase can be used in the kinetic resolution or enantioconvergent hydrolysis of racemic epoxides for prepare optically pure epoxides or vicinal diols, which has broad application prospects. To improve the enantioselectivity of Aspergillus usamii epoxide hydrolase (AuEH2) towards racemic p-methylphenyl glycidyl ether (rac-pMPGE). According to the protein-ligand finger-print (IFP) in the molecular dynamics simulation, the key residue site A250 with the highest interaction frequency towards (R)-pMPGE was selected, and then replaced by other 19 residues by site-directed mutagenesis. A mutant with the improved enantioselectivity was obtained and purified by affinity chromatography. Furthermore, the kinetic parameters and regioselectivity coefficients towards (R)-and (S)-pMPGE of the purified mutant were measured, respectively, and the recombinant E. coli whole cells was applied to the kinetic resolution of rac-pMPGE. The mutant AuEH2A250H possessed the highest E value of 38.4, which was remarkably higher than that of AuEH2 (12.7). The specific activity of E. coli/aueh2A250H was determined to be 51.9U/g wet cells. The kcat/Km for (S)-pMPGE of the purified AuEH2A250H was increased from 10.0mmol/(L·s) to 12.8mmol/(L·s), while decreased from 1.13mmol/(L·s) to 0.35mmol/(L·s) for (R)-pMPGE. Furthermore, using the whole cells of E. coli/aueh2A250H as biocatalyst, the kinetic resolution of 20mmol/L rac-pMPGE was performed at 25℃ for 1h, obtaining (R)-pMPGE with > 99% ees and 40.7% yield. The results indicated that the mutations at A250 played an essential role in regulating the activity and enantioselectivity of AuEH2. The mutant with the improvement of enantioselectivity of AuEH2, which has potential for industrial application on the preparation of (R)-pMPGE.
Immunotherapy is one of the effective methods to prevent and treat diseases. In recent years, tumor immunotherapy has become a new treatment method and related anti-tumor vaccines have been proved to be effective in the treatment of a variety of tumors. However, in the design of tumor vaccine, the problems of weak immunogenicity and low response rate of tumor antigens are a major challenge. The addition of adjuvants provides a new way to solve the problem. Immune adjuvants play an important role in improving the immunogenicity of tumor antigens and activating adaptive immune response. In order to understand the development and research status of immune adjuvants in recent years, the adjuvants commonly used in tumor vaccine were reviewed, and the mechanism of adjuvant on immune system was summarized, which provided help for vaccine design strategy in future.
Exosomes are extracellular vesicles which released upon fusion of multivesicular bodies with the cytoplasmic membrane. Exosomes were shown to contain functional proteins, lipids, and nucleic acids derived from secretory cells that mediating cell-to-cell communications and hence playing important roles in the physiology of the healthy and diseased organism. At present, the investigation on the mechanism of action of exosomes in viral infection and the potential markers of exosomes as a diagnosis and treatment of viral infection are still in their infancy. First described the composition and biogenesis of exosomes, and then focused on the mechanism of action of exosomes in viral infection, especially its role in immune regulation, and finally explored the diagnoses of exomes as a viral infection. And the potential for therapeutic potential markers and their application prospects.
Fat, as the largest secretory organ in the body, can regulate other tissues and organs by releasing hormones and cytokines. In recent years, studies have found that adipose tissue can release the form of exosomes, transmit signals to other tissues and organs through the circulation of body fluids, and regulate the physiological functions of its target organs, such as regulating liver fat deposition, muscle glycogen synthesis, angiogenesis, and damage repair. Such functions, for different target organs, exosomes will have different effects. The homeostasis of the body is the result of interactions between tissues, and the discovery of exosomes provides a stable material basis for the interaction of adipose tissue with other tissues, and the role of fat exosomes still has many unknown effects. The identification of adipose tissue exosomes and the interaction of adipose exosomes on the liver, muscle, and other tissues and organs from recent experimental reports were reviewed, provide a theoretical basis for the study of fat exosomes, and better explore the mysteries of life.
Fibroblast growth factor 5 (FGF5) is a member of the fibroblast growth factor family (FGFs) and is expressed in mammalian hair follicles, nervous system, testis and other parts of the embryo and during embryonic development. The study found that FGF5 has a wide range of biological activities, such as an important regulator of hair growth, its genetic mutation will lead to abnormal hair growth; as a mitogen plays an important role in stem cell proliferation, angiogenesis and limb muscle development; It has important biological functions in hypertension and tumors. At present, the function and mechanism of action of FGF5 in various diseases need further study, but its biological effects in hair growth, stem cell proliferation and cardiovascular diseases have great significance and clinical application value. The research progress of FGF5 in recent years, systematically expounds the related mechanism of FGF5 in hair growth, stem cell proliferation and differentiation, cardiovascular disease and cancer, and provides further research on the role and development of FGF5 in disease treatment were summarized.
In flowering plants, cytosine C at hundreds of sites is usually converted into uracil U by deamination in mitochondrials and chloroplasts, which is a regulation mechanism of genetic information at the transcriptional level. RNA editing requires protein-protein or protein-RNA interaction to assemble into a complex"editosome" for recognition at special sites in plant organelles. Reachers have found that plant RNA editing is regulated by editosome pentapeptide repeat PPR proteins, and nor-PPR protein including that RNA editing factor interacting proteins/multiple organellar RNA editing factor (RIP/MORF), organelle RNA recognition motif(ORRM) proteins, organelle zinc-finger (OZ) proteins and others. These explain that plant RNA editing not only has a high diversity in PPR specific factors, but also nor-PPR protein components. The absence of RNA editing will cause adverse effects such as stunted growth, development of plants and fruit ripening. The most recent progress in the field and discuss the editosomes for the evolution of RNA editing and for futue findings were systematically summarized.
As a biosynthetic molecule, DNA hydrogels are equipped with specificity, biodegradability and molecular recognition belonging to DNA molecule and high hydrophilicity belonging to hydrogels. Stimuli-responsive DNA hydrogels are mainly prepared by response behaviors of DNA branch structure formed by complementary pairing of bases of DNA sequences or i-motifs formed by DNA special sequences of many functional nucleic acid, T-A·T or C-G·C + triplexes and G-quadruplexes in environment triggers. Recently, stimuli-responsive DNA hydrogels possess broad applications of biosensors, bioimaging, drug delivery, biomaterials etc. with unique response property in single triggers such as temperature, pH, light, metal ions etc. and multi-triggers such as photothermal, metal ions-organics, temperature-pH. The origins of DNA molecule, classification, formation and characterization of stimuli-responsive DNA Hydrogels are developed. Response behaviors to environments and applications of stimuli-responsive DNA hydrogels are reviewed. The current research hotspots of stimuli-responsive DNA hydrogels are summarized. The development trend of stimuli-responsive DNA hydrogels in the future is predicted.
Xylitol is widely used in various fields because of its special physical and chemical properties, the global demand is increasing day by day. At present, the industrial production method of xylitol is produced by pure D-xylose under high temperature and pressure by chemical catalysis. There are many problems such as high requirements for raw materials, high energy consumption, rigorous conditions and heavy pollution. Biological fermentation technology can produce xylitol from low-cost crop waste fermented by strains. It has attracted much attention because of its wide source of raw materials, low energy consumption, mild condition and friendly environment, is a potentially attractive alternative to chemical processes. However, due to the low content of xylitol in fermentation products, the route of microbial fermentation to prepare xylitol has not been practiced in industry. The influencing factors in the process of bio-synthesis of xylitol and the possible technology of strain modification were reviewed. The challenges and prospects of bioproduction of xylitol were presented, and the research direction of biosynthesis of xylitol was prospected.
Glycosylation is an important post-translational modification, which has important effects on expression regulation, folding, secretion and function of the proteins. Glycosylated modification mainly exists in eukaryotes, however it is found that some prokaryotes also possess that modification. Depending on the type of amino acid linked to the sugar chain, glycosylation can be divided into N-glycosylation and O-glycosylation. Enzymes are highly specific and efficient biocatalysts produced by living cells, which are proteins or RNA in chemical nature. The role of glycosylation in the internal structure and external function of enzymes has been a research hotspot in recent years, especially the influence on biocatalytic performance and stability. It has attracted much attention due to its far-reaching significance on the design and modification of enzymes. To understand the mechanism of influence made by glycosylation modification on enzyme structure and biocatalytic activity can provide ideas and directions for rational/semi-rational design and modification of enzyme molecules. X-ray crystallography is one of the main methods to study the structures of glycosylated enzymes, which combine X-ray diffraction and protein crystallography. However, complex sugar chains cause chemical and structural heterogeneity of glycosylated enzymes, which hinders crystal formation and crystal diffraction. Hydrolysis treatment with glycosidase, introduction of glycosyltransferase inhibitors and optimization of heterologous expression system are all important strategies to improve the crystallizability of glycosylated enzymes. These methods can improve the homogeneity of glycosylated enzymes while avoiding the damage to stability and catalytic activity of those.
The old yellow enzyme family (OYEs) is a large family which widely distributed and capable of catalyzing the asymmetric reduction of olefin compounds. It can be used in the preparation of various chiral compounds. The system classification and catalytic reaction types of OYEs were analyzed. Meanwhile, protein engineering methods on the modification of the stability, activity and switching the substrate specificity of OYEs are also discussed. Some clues for further study the catalytic mechanism of OYEs and lays the foundation for further expanding the industrial application of OYEs were given.
