Objective: To investigate the effects of phospholipase C epsilon on serine/glycine metabolism and cell proliferation in prostate cancer cells.Methods: Lentivirus and plasmid were transfected into LNCAP and PC3 cells. The expression of YAP, serine/glycine producing enzyme (PSAT1,PSPH,SHMT2) and proliferation-related genes (Cyclin D1,PCNA) were detected by q-PCR and Western blot. The cloning formation experiment and MTT assays were used to detect the clone formation rate and cell proliferation activity.Results: (1) Infection with LV-shPLCε significantly down-regulated the mRNA and protein levels of PLCε,YAP,serine/glycine producing enzymes (PSAT1,PSPH,SHMT2) and proliferation genes (Cyclin D1,PCNA) in prostate cancer cells LNCAP and PC3. At the same time, it inhibits the clone formation ability and proliferative activity of LNCAP and PC3 cells. (2) After adding over-expressing YAP plasmid to shPLCε group, YAP,serine/glycine producing enzymes and proliferation genes were significantly reversed. but the results of the interference with the down YAP plasmid were reversed.Conclusion: shPLCε inhibits the serine/glycine metabolism and proliferation in prostate cancer cells by down-regulating the expression of YAP.
Objective: To invest igate the effect of TAGLN on the biological behavior of HBV-positive hepatocellular carcinoma cell line HepG2.2.15 and its underlying mechanism.Methods: Immunohistochemistry and Western blot were used to detect the expression levels of TAGLN in HBV-positive and HBV-negative hepatocellular carcinoma tissues and cells; HepG2.2.15 cells were infected with TAGLN interfering lentiviruses, and stable expression cell lines were screened by purinomycin. Then transfection efficiency was confirmed by Western blot; CCK-8 and clone formation assay were used to detect the proliferation of HepG2.2.15 cells. While cell migration and invasion capabilities were determined by Transwell assay. The expression levels of PI3K, p-PI3K, AKT and p-AKT were examined by Western blot.Results: The expression of TAGLN in HBV-positive hepatocellular carcinoma tissues and cells was higher than that in HBV-negative hepatocellular carcinoma tissues and cells(P<0.01); Knockdown of TAGLN could effectively inhibit cell proliferation, migration and invasion (P<0.01), as well as down-regulate the expression of PI3K, AKT(P<0.01), p-PI3K and p-AKT (P<0.05).Conclusion: HBV infection can increase the expression of TAGLN in hepatocellular carcinoma. After interfering with TAGLN expression, the proliferation, cloning, migration and invasion of HepG2.2.15 cells decreased, which may be related to the decrease of PI3K and AKT expression.
Resveratrol synthetase (RS), an important enzyme of chalcone synthase gene super family, catalyzes resveratrol production in plants. Resveratrol is a non-flavonoid polyphenol metabolite, which is a phytoalexin produced by plants under both biotic and abiotic stress. It has been proved that resveratrol has a variety of physiological activities.The CDS sequence annotated as CHS gene was obtained from the database of transcriptome, and the full length gene sequence was cloned using Caragana intermedia cDNA as template. Sequence, phylogenetic analysis and characteristic study of transgenic Arabidopsis indicated that the gene was a RS gene, then it was named CiRS. The expression of CiRS was induced by drought, NaCl and ultraviolet (UV) stresses as revealed by quantitative real-time PCR (qRT-PCR). Heterologous expression of CiRS gene inhibited the expression of endogenous AtCHS gene in transgenic Arabidopsis, meanwhile the antimicrobial activity of transgenic lines was stronger than that of wild type. All these results confirmed that the CiRS of C. intermedia played roles in transgenic Arabidopsis.
Weeds and insect pests are two important biotic stresses to result in yield loss in rice, and breeding bioengineered rice with herbicide resistance and insect resistance is one of the effective ways to solve these two problems. And for that, the glyphosate resistance and Lepidopteran resistance as well as molecular and some agronomic characteristics of transgenic line E1C608 in rice were identified. Results of molecular identification showed that three independent transformation events were obtained, in which one single copy insertion event was named as E1C608-3. The transformant E1C608-3 of T2 generation was used to detect exogenous protein contents in root, stem and leaf at tillering stage by ELISA. The protein contents of EPSPS and CRY1C in different organs of E1C608-3 were significantly different both in descending order of leaf>stem>oot (P<0.01), ranged from120.16μg/g to 1 223.28μg/g and from 1.23μg/g to 8.72μg/g, respectively. The glyphosate tolerable concentration at seedling stage of E1C608-3 in T3 generation reached at least 16g/L, which was sixteen times higher than that of the transformation receptor R608. The larva mortality of rice leaf roller fed on leaves of E1C608-3 in T3 generation for five days was 95.56%, that showed the E1C608-3 with an excellent resistance to Lepidopteran insects. The data suggested that some agronomic traits were significantly different between E1C608-3 in T4 generation and R608 (P<0.01), but all of the variations were within the variation range of natural germplasm resource in rice. Overall, a novel germplasm with glyphosate resistance and Lepidopteran resistance was verified to be developed in rice.
Members of the G protein-coupled receptor (GPCR) family play important roles in the sensing of extracellular signals. Ste2 is one of the three GPCR proteins in the budding yeast, Saccharomyces cerevisiae. In the past years, extensive efforts have focused on how the function and expression of Ste2 are affected by various mutations. However, little is known about the mechanisms dictating its proper subcellular localization. To this end, a series of mutants containing deletions or substitutions in the N-terminus, C-terminus, transmembrane domains, intra/extracellular loops are constructed. The subcellular localization of wild-type and mutant Ste2 proteins are observed by fluorescent microscopy, in combination with a set of organelle markers, to determine their localization. Wild-type Ste2 is primarily targeted to the plasma membrane and vacuolar lumen. Deletion of the C-terminus eliminates the vascular signal, targeting the protein to the plasma membrane and endoplasmic reticulum instead. The result is similar when the C-terminus is substituted by the corresponding regions from ORI7 and OR17-40, two mammalian GPCRs. When the N-terminus, first extracellular loop (EL1), the second extracellular loop (EL2) or the third intracellular loop (IL3) is substituted, plasma membrane targeting of Ste2 is substantially attenuated or eliminated. Some of these mutants accumulate on intracellular punctate structures. These results suggest that the N-terminus, EL1, EL2 and IL3 regions contain potential sorting signals regulating the transport of Ste2 to the plasma membrane, and that the C-terminus contains signals for its targeting to the vacuole. The present work provides new insights towards understanding the mechanisms governing GPCR protein subcellular localization.
Objective: The molecular mechanism of cytosolic DNA induced cellular glucose metabolic response was aimed to unravel.Methods: (1) The nucleus and cytoplasm were separated by fractionation, and the protein bound to cytosolic DNA (ISD) was isolated using biotin-avidin affinity chromatography. The differentially expressed protein, AUF1 was identified by silver staining followed by mass spectrometry analysis or directly by complex-mass spectrometry analysis. The interaction between AUF1 and ISD was verified by pull-down assay. (2) ATP assay and CCK8 analysis were performed to evaluate the cytosolic DNA induced cellular glucose metabolic response in wildtype and AUF1 knockout cells, which was generated by CRISPR/Cas9 technology. (3) The mRNA expression of glucose transporters GLUTs and key enzymes in the process of glucose metabolism were detected by semi-quantitative PCR in four types of cells: wild type HEK293 cells, AUF1 knockout HEK293 cells, and AUF1 knockout cells reconstituted with AUF1 or empty vector as controls. GLUT3 was identified as one of the downstream effectors of AUF1. Real-time PCR was also performed to verify the results. (4) GLUT3 mRNA under the stimulation of cytosolic DNA was analyzed by semi-quantitative and real-time PCR.Results: (1) Both mass-spectrometry analyses showed that AUF1 could bind to ISD. In vitro binding assays also confirmed that both GST-AUF1 expressed in prokaryotic cells and GFP-AUF1 expressed in eukaryotic cells could bind to single-stranded and double-stranded ISDs. (2) Upon cytosolic DNA stimulation, intracellular ATP levels and reductive capabilities of AUF1-/- HEK293 cells were higher than wild-type cells. It suggested that the glucose metabolism in AUF1 knockout cells is not inhibited by cytosolic DNA stimulation, and AUF1 may be involved in cytosolic DNA induced cellular glucose metabolic response. (3) Semi-quantitative PCR analysis showed that GLUT3 mRNA expression was significantly reduced in AUF1 knockout cells, while there were no significant differences among other GLUT family members and metabolic enzymes. Real-time PCR also confirmed the above phenomena, suggesting that AUF1 may regulate glucose metabolism by stabilizing GLUT3 mRNA. (4) Both single-stranded and double-stranded ISD stimulation lead to a decrease in GLUT3 mRNA expression, suggested that GLUT3 might be a downstream effector in the regulation of glucose metabolism upon cytosolic DNA stimulation.Conclusions: AUF1 can bind to cytosolic DNA, and participate in the cytosolic DNA induced Glucose metabolic response, potentially through regulating the stability of GLUT3 mRNA.
A novel nucleic acid detection method, based on the xMAP (flexible multi-analyte profiling) technology platform, was developed for high-throughput and simultaneous identification of fox and mink ingredients in products of animal origin. A duplex xMAP assay was established, the primers and probes were designed targeting a fox mitochondrial D-loop gene or a mink mitochondrial cytochrome b gene. The probes were incorporated with locked nucleic acids to improve detection efficiency. The assay accurately identified fox and mink DNA, without cross reactions with DNA samples of eighteen nontarget animal species. The LOD on purified fox and mink DNA was evaluated as 2.8pg/μl and 0.9pg/μl, respectively. The detection sensitivity on samples of experimental meat mixtures was demonstrated to be 0.05% (m/m). The assay successfully detected 32 mocked positive DNA sample of food and feeds, which each was made by adding fox DNA or mink DNA at 1% (V/V) proportion of the same concentration. In conclusion, the duplex xMAP assay provided rapid identification of fox and mink ingredients with high specificity and high sensitivity. The technique is suitable to be applied in food and feeds quality assurance systems and safety inspections.
Nitrile hydratase are enzymes that catalyze the conversion of nitriles to their corresponding amides. There are many problems in the catalytic process of free cells containing nitrile hydratases, such as low cell utilization rate and the high cost of later separation, which can be solved effectively by immobilization of cells. Several commonly embedding immobilization methods by evaluating the reaction batches and the activity recovery rate of immobilized enzymes with nitrile hydratase containing recombinant E.coli cells as the research object to find a suitable immobilization method were compared.The results showed that DA-F127 was the most suitable material for immobilization of recombinant E. coli cells. When the concentration of DA-F127 was 15%, the UV irradiation distance was 20cm, the irradiation time was 6min, and the cell content was 20mg/g carrier, the recovery rate of enzyme activity is 89.74%, and it could be reused up for 9 batches to covert 150g/L 3-cyanopyridine. The conversion rate of the ninth batch could reach to 98.26%. The nicotinamide yield was increased by 12 times compared with the same amount of free cell catalysis.
In order to further simplify the genetic operation process of actinomycetes and shorten the screening cycle of the recombinant strains, a counter selectable marker gene —— uracil phosphoribosyl transferase gene (upp) was introduced into the genetic operation of Streptomyces fungicidicus ATCC 21013. By deletion of the upp gene in the wild type strain and construction of a suicide vector that carries the upp gene expression cassette, a markerless inframe deletion system was developed, and the StrR gene in S. fungicidicus genome was successfully knocked out after vector integration, plasmid excision and counterselection. The introduction of upp gene shortened the average screening cycle of Streptomyces recombinant strains by about 2 weeks, and further reduced the probability of false positive recombinant strains. The simplicity of this system should make it adaptable for continuous markerless gene deletion in other actinomycetes, so it is worth further promotion and application.
Gene editing is a new technology of precise gene modification which provides a powerful tool for gene function analyses.Currently,these methods such as zinc-finger nuclease (ZFN),transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) are available for researchers to operate target genes. Also fields like gene function analyses and medical therapy have been innovated. The types and principles of gene editing technology are summarized.The research progress of CRISPR gene editing technology in the treatment of diseases was highlighted. In the end,the future research of gene editing technology is prospected.
Aptamers are single-stranded DNA or RNA who bind to their targets with high specificity and affinity obtained by selection from a library through SELEX. At present, there are only a few aptamers with high affinity and high specificity to low molecular weight targets all over the world. The aptamers against low molecular weight targets are difficult to be screened. Moreover, it is difficult to determine the affinity of low molecular weight targets binding with their candidate aptamers. Affinity characterization is the key step to determine whether the aptamer screening is successful or not. The existing affinity characterization methods for low molecular weight targets and their corresponding aptamers are summarized, including nanogold colorimetry, isothermal drop calorimetry, surface plasmon resonance, circular dichroism, quartz crystal microbalance, microscale thermophoresis and SYBR Green I dye detection. The advantages and disadvantages of these methods and suggestions for improvement are also discussed in order to improve the efficiency of aptamer characterization.
As the increasingly develepoment of mRNA stability and delivery systems, the mRNA vaccine has made rapid progress in the individualized tumor vaccine in recent years. Due to its simple production process, expression the antigens in cells, and security features is superior to DNA vaccine, mRNA is a new form of vaccine promising alternative to attenuated and inactivated vaccine and protein vaccine.In order to understand the development and research status of global mRNA vaccine, the molecular design, delivery system and clinical research status of mRNA vaccine were analyzed and summarized, providing reference for the subsequent development and research of mRNA vaccine.
The construction of mutant library is a key step in the process of directed evolution. It mainly utilizes the system of natural or synthetic molecular technique to generate the diversity of the nucleic acid molecular libraries. It also provides large genetic gene pool for the preparation and screening of protease, polypeptide and artificial antibodies with specific properties. Furthermore, it can be used in the study and selection of genetic element in synthetic biology, providing power to highly industrialized production of biological products. With the deepening of the research on the construction technology of mutant libraries, various library construction strategies have been developed and widely applied in the fields of bioenergy, biochemistry, biomedicine, biological reagent and food industry. However, the strategies of library construction in the directed evolution are different, and the core methods of various mutant library construction technologies are constantly innovated. Here it mainly elucidates the leading technologies of synthesizing multiplex libraries for the laboratory in recent years, and prospects for the development of library construction technology in the direction of automation and intellectualization.
Plant isoquinoline alkaloids (PIAs) include morphine, codeine, galantamine, berberine and other pharmaceutically active products. Currently, most high-value PIAs are extracted from plants, which is limited by low concentration in nature, seasonal production and extraction methods. Microbial biosynjournal provides an alternative way to gain PIAs at low cost, however, the long and complex biosynjournal pathways of PIAs bring it many challenges. With the development of synthetic biology and biotechnology, many progresses are made in the pathway elucidations and enzyme identifications, which make it possible to achieve the biosynjournal of PIAs in heterologous microbes. Recent advances in the PIAs pathway elucidations and metabolic engineering in heterologous microbes are reviewed, and current challenges as well as future perspectives are discussed.
