Objective: To explore the effect of miR-290-295, the most abundantly expressed microRNA cluster in stem cells, as a whole on somatic cell reprogramming. Methods: The miR-290-295 cluster was overexpressed into mouse somatic cells using retroviral vectors to explore whether it promotes the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and the effect on cell function through this process. Results: The overexpression of miR-290-295 cluster could significantly improve the efficiency of mouse somatic cell reprogramming in the traditional induction system of three factors (Sox2, Klf4 and Oct4). Overexpression of miR-290-295 cluster could not only promote the up-regulation of pluripotency marker genes and the down-regulation of somatic marker genes during reprogramming, but also the expression of mesenchymal-epithelial transition (MET) marker genes and cell proliferation related genes. Conclusion: miR-290-295 promotes the reprogramming of mouse somatic cells. Our findings are helpful to understand the RNA regulatory mechanism in stem cell pluripotency and reprogramming, and provide a new perspective for the development of new induction systems.
Objective: To analyze the catalytic activity of GSK-3β for tau protein phosphorylation in vitro and the effect of phosphorylation modification on tau aggregation and cytotoxicity. Methods: Recombinant human glycogen synthetic kinase-3β (GSK-3β) was expressed and purified by prokaryotic and baculovirus expression systems. GSK-3β expression vectors with C-terminal tag were constructed. The proteins were purified through nickel affinity chromatography, and the protein concentrations were determined by BCA kit. SDS-PAGE Coomassie bright blue staining was used to analyze the purities of proteins. The immunoreactivity of recombinant GSK-3β protein was determined by Western blot. Protein phosphorylation was conducted through GSK-3β and recombinant human tau441 incubation in Tris-HCl solution. The concentrations of enzyme and adenosine triphosphate (ATP) were optimized. The phosphorylation of recombinant human tau441 was detected by liquid chromatograph mass spectrometer (LC-MS) and dot blot. The aggregation of phosphorylation products were determined by negative staining transmission electron microscopy (TEM) and thioflavin T (ThT) binding assay. Results: The data of SDS-PAGE showed that the apparent molecular weight of recombinant human GSK-3β protein expressed by prokaryotic virus and baculovirus was about 50 kDa and the purity of recombinant human GSK-3β protein was 86% and 81%, respectively. Western blot showed signal bands in corresponding positions. LC-MS analysis showed that 23 sites of tau protein were phosphorylated after GSK-3β treatment. Dot blot showed that rabbit anti-pT181 serum, pT217 and pS404 antibodies (with priority recognition of tau181, tau217 and tau404, respectively) recognized tau441 phosphorylated in vitro. The optimal concentrations of tau, GSK-3β and ATP was 1 μmol/L, 5 μmol/L and 3.2 mmol/L, respectively. The phosphorylation effect of GSK-3β prepared in this study on pT217 was significantly stronger than that of imported products (P<0.05). TEM images showed that fibers appeared in phosphorylated tau441 at 5 d and matured at 14 d. However, no obvious fiber structure was found in the tau441 group. Accordingly, ThT binding assay showed that the fluorescence value of phosphorylated products increased (P<0.05). Moreover, the cytotoxicity of phosphorylated products increased (P<0.05). Conclusion: Recombinant human GSK-3β-His and GSK-3β-His-Bac1 proteins were successfully prepared. These two proteins have the function of catalyzing in vitro phosphorylation of tau protein at amino acids 181, 217 and 404, which provided technical support for related basic research on Alzheimer’s disease.
Objective: To investigate the effect of miR-296-3p on granulosa cell apoptosis in rats with polycystic ovary syndrome (PCOS). Methods: The levels of estradiol (E2), progesterone (P) and testosterone (T) in serum of PCOS rats were detected by automatic chemiluminescence immunoassay; HE staining was used to detect the pathological changes of the ovary; TUNEL was used to detect apoptosis in the ovary; The expression level of miR-296-3p and PRKCA in the ovary was detected by qPCR; Ovarian granulosa cells (GCs) were divided into miR-NC, miR-296-3p mimic, miR-296-3p inhibitor, miR-296-3p mimic+Vector, miR-296-3p mimic+PRKCA and miR-296-3p mimic+PRKCA+NF-κB groups after transfection of various plasmids, and untreated GCs were used as control group. The expression level of p38, p-p38 and NF-κB was detected by Western blot. StarBase website and double luciferase report test predicted and verified the targeting relationship between miR-296-3p and PRKCA, respectively. Flow cytometry was used to detect cell apoptosis rate. Results: Compared with the control group, the levels of E2, P and T in the ovaries of PCOS rats increased; The proportion of apoptosis increased; The expression of miR-296-3p increased. miR-296-3p has a targeting relationship with PRKCA, and there are binding sites in the 3'UTR region. Compared with miR-NC group, the expression level of PRKCA mRNA in miR-296-3p mimic group was significantly decreased, and p-p38/p38 and NF-κB expression was up-regulated. Compared with miR-NC group, the expression level of PRKCA mRNA in miR-296-3p inhibitor group was significantly increased, and p-p38/p38 and NF-κB expression was down-regulated. Compared with the Mir-NC group, the percentage of apoptosis of miR-296-3p mimic and miR-296-3p mimic+Vector groups was significantly up-regulated. Compared with miR-296-3p mimic and miR-29-3p mimic+Vector, the percentage of apoptosis of ovarian granulocyte cells in miR-296-3p mimic+PRKCA group was significantly down-regulated. Compared with miR-296-3p mimic+PRKCA+NF-κB group, the percentage of apoptosis of ovarian granulosa cells in miR-296-3p mimic+PRKCA+NF-κB group was significantly up-regulated. Conclusion: MiR-296-3p activates p38 MAPK/NF-κB signal pathway by targeting PRKCA and mediating granulosa cell apoptosis in rats with polycystic ovary syndrome.
Objective: It is reported that NtASAT1 (Nicotiana tabacum acylsugar acyltransferase) from tobacco can transform sucrose and short branched chain fatty acids to sucrose monoester. Thus, we tried to use the prokaryotic expression system of Escherichia coli to analyze the expression and purification condition of NtASAT1 and further verify the function of purified NtASAT1. Methods: First, the physical and chemical properties, secondary structure and homology of tobacco NtASAT1 were analyzed by bioinformatics software. Then, the gene NtASAT1 was cloned from the cDNA of tobacco glandular hairs and constructed into the expression vector so as to study its expression in BL21 (DE 3). Finally, NtASAT1 was purified by nickel column and the activity of purified target protein was then analyzed by enzyme reaction. The product of the enzymatic reaction was analyzed by liquid chromatography-mass spectrometry (LC-MS). Results: NtASAT1, which was truncated by 93 amino acids at the C-terminal, could be expressed in BL21 (DE3). Most of the expressed protein existed in an insoluble state and different concentrations of inducer, induction time and induction temperature had no obvious effect on the soluble expression of the protein trNtASAT1. The target protein was purified by nickel column and after enzyme reaction by adding substrate, the product sucrose monoester could be detected by LC-MS. Conclusion: The recombinant protein trNtASAT1 was cloned and purified and the enzymatic product sucrose monoester was detected by LC-MS, which proved that the purified NtASAT1 was functional. This study laid the foundation for purification and further application of enzyme ASAT in industry.
Objective: To explore the mechanism for enhanced cAMP fermentation production by polyphosphates, Arthrobacter sp. CCTCC 2013431 culture was carried out under low-polyphosphates addition condition as the starting strain. Methods: Fermentations with/without hexametaphosphate addition were conducted in a 7 L bioreactor and the fermentation performance, global gene transcriptome, key enzymes activities together with important metabolites levels were analyzed systematically. Results: With 2 g/L-broth sodium hexametaphosphate added at 24 h, cAMP concentration reached 3.64 g/L with an increment of 33.82% higher than that of control group and the fermentation performance was also promoted obviously. Transcriptome analysis showed that 227 genes were up-regulated significantly and 265 genes were down-regulated significantly due to the addition of hexametaphosphate. For glycometabolism, the transcription levels of key enzyme genes in pentose phosphate pathway and cAMP synthesis pathway were enhanced significantly and for energy metabolism the transcription levels of complex Ⅲ, complex Ⅳ as well as F0F1-ATPase in electron transport chain and polyphosphate kinase gene were also increased significantly by which sufficient carbon skeleton and ATP were provided for cAMP biosynthesis. In addition, transcription levels of reductase genes, such as thioredoxin, catalase and CLP protease, were also increased significantly whereby intracellular redox balance was maintained conducive to cell metabolism and product synthesis. Finally, the activities of pyruvate kinase, 6-phosphoglucose dehydrogenase, adenylosuccinate synthetase, adenylate cyclase, catalase, polyphosphate kinase and intracellular ROS, ATP and NADPH levels under different fermentation conditions were measured to further support the transcriptome analysis results. Conclusion: Sodium hexametaphosphate addition enhanced the carbon flux distribution in pentose phosphate pathway and cAMP synthesis pathway and energy metabolism for ATP synthesis. At the same time, intracellular redox balance was also maintained. Furthermore, cAMP fermentation synthesis and accumulation was promoted significantly.
Objective: Tth DNA polymerase (Tth pol) is a thermostable polymerase with both DNA polymerase and reverse transcriptase activities. To control the non-specific amplification in the one-step reverse transcription-quantitative real-time PCR (RT-qPCR), the aptamers that inhibit the activity of Tth pol mutant at moderate temperature were screened to perform warm-start one-step RT-qPCR. Methods: Based on the Mn2+-independent Tth pol mutant obtained in previous study, the effects of four DNA aptamers and their corresponding RNA aptamers on the inhibition of DNA polymerase and reverse transcriptase activity of this Tth pol mutant and the dissociation temperature were examined. The binding poses between these aptamers and Tth pol mutant were simulated by molecular docking. Results: Two aptamers TQ21-11 and TQ21-11-RNA can block two distinct activities of Tth pol at 40℃ with inhibition rates of over 97% and all of them can be almost completely dissociated at 50℃. Both aptamers were validated for warm-start one-step RT-qPCR, with lower non-specific amplification of TQ21-11-RNA. Conclusion: The aptamers TQ21-11 and TQ21-11-RNA were able to effectively block the activity of Tth pol mutant at moderate temperature and perform warm-start one-step RT-qPCR well.
As a common human malignant tumor, the pathogenesis of hepatocellular carcinoma (HCC) has not been completely clarified. Wnt signaling pathway is related to a variety of pathophysiological processes in human body, among which the carcinogenesis and the development of HCC may be closely related to the classic Wnt/β-catenin signaling pathway. Wnt/β-catenin regulates HCC by expressing cancer-related genes, activating hepatic stellate cells, regulating liver stem cell behavior, and promoting the invasion and metastasis of cancer cells. The role of Wnt/β-catein signaling pathway in HCC is discussed.
Traditional bone tissue engineering scaffold materials have disadvantages, such as insufficient strength and low bioactivity. In recent years, nanomaterials have shown many unique advantages in bone regeneration. Graphene oxide (GO), an oxidized form of graphene, is a representative two-dimensional nanomaterial. Many studies have shown that GO has excellent mechanical properties, good biocompatibility, large specific surface area and easy modification.GO can not only directly promote the adhesion, proliferation and differentiation of stem cells, but also improve the mechanical properties, biological activity, antibacterial ability and immunomodulatory ability of traditional scaffolds. GO-based composite materials are expected to become ideal scaffolds for bone regeneration. This paper first describes the physicochemical properties, biocompatibility, biodegradation and clearance of GO. Then,the latest applications of GO as coatings, controlled release agents and composite scaffolds in bone tissue engineering were summarized. At last, the advantages and challenges of GO are analyzed, and the future research direction is forecasted.
Stem cell transplantation has been regarded as an attractive alternative therapy in medical research and clinical trials. It is confirmed by numerous experimental model studies that stem cell transplantation can repair damaged or degraded tissues and restore their function. However, immune rejection, ethical barriers and tumorigenicity are still the difficulties in the practical applications of stem cell therapy. In recent years, perinatal stem cells have been paid increasing attention by researchers as a potential source of cells to solve the above-mentioned problems. Compared with other stem cells, human amniotic stem cells (hAMSCs) show significant advantages in these aspects. Animal experiments have found that hAMSCs have high differentiation potential and immunomodulatory activity, demonstrating great potential for the treatment of gynecological diseases, neurological diseases, kidney diseases, lung diseases, skin diseases, diabetes, cancer and other diseases. At present, with the progress of science and to meet urgent clinical needs, the clinical application of hAMSCs has gradually broken through the limitations of traditional treatment. Many clinical studies have been registered to study the effectiveness and safety of hAMSCs, and some studies have been completed as planned, which has important guiding significance for clinical practice. Therefore, this article reviews the biological research progress and application potential of hAMSCs, in order to provide theoretical basis for experimental research and clinical application of hAMSCs.
Optogenetics is a new technology that combines genetics technology and light control technology to control biological processes with light. Based on the strategy of combining optogenetics and synthetic biology methods, researchers combined light as a sensing module with existing gene modules in organisms to form a new gene circuit, and designed a system that can dynamically regulate gene expression through light signals. As a new type of dynamic regulation switch with low cost, low toxicity and high flexibility, it has not only been applied in disease diagnosis, material synthesis and other fields, but also greatly promoted the progress of microbial metabolism and helped solve energy shortage problems. Since photoreceptors are indispensable components in optogenetics technology, this paper introduce several light-regulated systems used to control gene expression according to the photosensitive properties of different photoreceptors, and focus on their applications in regulating microbial system gene expression, metabolic pathways and drug delivery. Meanwhile, the potential problems and prospects of the application of photogenetics in synthetic biology were also discussed.
With the coming of the post genome era, simple and efficient gene editing tools and methods have played a critical role in studying the regulation of gene expression and the genetic modification of engineered strains. The counter-selection methods are much convenient and efficient for gene knockout, which are based on the principle of homologous recombination. Knockout or insert of one target gene is achieved by the loss or supplement of one selective genetic marker, which generally does not rely on the traditional antibiotics. To achieve genetic manipulation of more target genes, the same selected marker can be used repeatedly over the progress of successive manipulation rounds. Because of its versatility, simplicity, efficacy, and wide range of applications, the counter-selective system has been applied in many fields of research, such as biotechnology, genetic engineering, and fundamental and applied biology. In recent years, the types, mechanisms and applications of counter-selection marker genes in microbial gene knockout experiments are reviewed. This genome-editing technique has a valuable advantage that particularly attracts the scientist’s mind, as it allows genome editing in multiple biological systems, and supports great opportunities for the future development of microbial science and rapid remodeling of genes.
Various cofactors are required in the process of most enzyme-catalyzed reaction of cells. Cofactor balance is very important to maintain the homeostasis of biochemical reaction in cells. However, insufficient supply of cofactors will lead to the disorder of cell growth and compound production. In recent years, the key role of cofactors in the biochemical reaction process has attracted the attention of researchers, but most cofactors are expensive and have poor stability. These disadvantages have limited the potential application of cofactor engineering. The development of synthetic biology and metabolic engineering provides feasible solutions for the sustainable supply of cofactors, and multiple strategies to strengthen the supply of cofactors effectively promote the biosynthesis of target compounds. Among them, nicotinamide cofactors NAD(P)+ and NAD(P)H are the most common redox cofactors in the process of microbial metabolism. They are important electron receptors or donors in all organisms, promote synthesis and catabolism reactions, and play a decisive role in maintaining the dynamic balance of intracellular redox. Starting from the main sources of NAD(P)H and the influence of NAD(P)+/NAD(P)H balance in the synthesis of natural products, cofactor engineering strategies were reviewed from three different dimensions. This paper introduces the latest research progress and application of multiple cofactor regeneration strategies through metabolic pathway regulation, introduction of exogenous oxidoreductase, and protein engineering. The future development prospects of cofactor metabolic engineering in biosynthesis is also discussed.
As an important part of biocatalysis, whole-cell catalysis uses whole cells as catalysts, which eliminates the need of cell lysis and enzyme purification in the process of alcohol production, and thus greatly reduces the cost of alcohol production. At the same time, the cell wall components can protect the trapped enzyme from potentially harmful environmental factors, thus meeting the production requirements of low cost and high stability of catalysts. According to the advantages of cell catalysis technology and the current market application prospects, the suitable strains for producing alcohol substances, such as ethanol, diol and sugar alcohol, were discussed. The alcohol production by using cell catalysis technology in food and chemical industries was reviewed. Finally, the production strategy of increasing alcohol yield was also discussed.
