Objective:To demonstrate the role of metformin in oligodendrocyte precursor cell (OPC) differentiation and preliminarily discuss the molecular mechanism.Methods:OPC was directly isolated and purified by immune adsorption from the brain and identified using immunofluorescence. Firstly, the concentration of metformin was decided through cell viability assay. Then, the effects of metformin on OPC-differentiation related positive cells, the mRNA or protein level were analyzed by immunofluorescence, flow cytometry, qRT-PCR, and western blot.Results:High purity of primary cells were obtained. CCK8 assay showed that there is no significant toxicity of metformin (<100 μmol/L) on cell viability. Moreover, the significant increasement of PDGFRα+OLIG2+ and MBP+ cells, up-regulation levels of Mag, Olig2, Mbp and Sox10 mRNA and OLIG2, MBP protein were detected in OPC after metformin treatment. Mechanically, compared to the control group, RAS, p-MEK and p-ERK proteins were significantly increased after metformin treatment for 5min in Oli-neu cells and OPC. Conclusion:Metformin promotes the differentiation of oligodendrocyte precursor cells through the RAS-MEK-ERK signaling pathway.
Objective:To investigate the expression of autophagy-related genes and the interaction of autophagy-related proteins in liver tissues of ATP7B-deficient (WD) mice, and to explore the possible mechanism of copper accumulation induced autophagy activation in liver.Methods:The liver copper content of 4 weeks and 12 weeks of WD mice was detected. RNA-sequencing of liver tissues was conducted, and the GO and KEGG pathways of differentially expressed genes were analyzed by bioinformatics. The expression of autophagy-related differentially expressed genes was detected by qRT-PCR and Western blot. GeneMANIA database was used to construct the protein-protein interaction network (PPI) which was related to these autophagy-related proteins, and functional annotation was carried out to analyze its autophagy-related biological function and protein interactions. The expression of autophagy-related proteins was inhibited to analyze its effect on autophagy.Results:Compared with wild-type mice, liver copper content of WD mice was significantly increased, and the copper accumulation led to changes in gene expression pattern. According to the GO database, the number of autophagy-related differential genes in WD mice was 8 at 4 weeks and 51 at 12 weeks. According to KEGG database, the number of autophagy-related differential genes was 5 at 4 weeks and 19 at 12 weeks, respectively. Nine genes, including Ulk1, Ddit4 and Plk3, were screened for qRT-PCR, and the quantitative results was basically consistent with the sequencing results. These autophagy-related proteins interact with each other through co-expression and co-localization. Western blot results showed that copper accumulation significantly increased the protein expressions of Ulk1, Plk3 and Park2, and resulted in autophagy. Inhibition of Ulk1, Plk3 and Park2 expression significantly down-regulated the level of autophagy.Conclusion:Copper accumulation at different stages of WD can regulate the expression of several autophagy-related genes in the liver, and the liver autophagy activation was induced by the interaction of autophagy-related proteins which could alleviate liver injury of WD.
hox genes encode a family of highly conserved transcription factors. Human HOXA1 mutation causes ABDS (athabascan brainstem dysgenesis syndrome), which leads to craniofacial bone deformity induced facial defect and paralysis. In this paper, zebrafish was used to study the functional mechanism of the homologous gene hoxa1a. Firstly, hoxa1a gene was edited by using CRISPR/Cas9 technology, which resulted in gene mutation. The T7E1 assay showed F0 digestion efficiency was 70% on average. Then F1 was screened and it was found that hoxa1a heterozygote generated 8 bp insertion and 7 bp deletion. Furthermore, the heterozygotes were crossed and hoxa1a homozygous F2 mutant was obtained, which was confirmed by sequencing. At 5 dpf, homozygous mutants of hoxa1a showed craniofacial dysplasia. The results of alcian blue cartilage staining and alizarin red hard bone staining demonstrated that the hoxa1a mutant had abnormal skull development, fracture of ethmoid plate, and defect of gill arch development. In this study, ABDS disease model in zebrafish was successfully constructed and the results indicate that hoxa1a mutation might cause abnormal craniofacial skeletal development, which lays a foundation for its mechanism study and provides a new idea for the pathogenesis of human ABDS disease.
Weighted gene co-expression network analysis (WGCNA) is a systematic biological method. It can be used to explore the relationship between genes and related traits by the identification of co-expressed modules and hub genes in the network. Sweetpotato [Ipomoea batatas (L.)Lam.] is one of the nutritious tuberous crops in the world. Purple-fleshed sweetpotato (PFSP) is a special variety of sweetpotato. In addition to the nutritional components of common sweetpotato, PFSP is also enriched in anthocyanin with antioxidant function that is beneficial to health. Therefore, the cultivation of PFSP with high anthocyanin content has always been the goal of scientists. Although some PFSP varieties have been bred by traditional hybridization breeding techniques, which has some disadvantages, such as long breeding cycle, labor consuming and low efficiency. Therefore, it is urgent to develop PFSP with high yield and quality by molecular design breeding. Studying on key genes related to anthocyanin biosynthesis of sweetpotato can shed light on molecular breeding of PFSP. In order to explore genes related to anthocyanin biosynthesis in sweetpotato, white-fleshed sweetpotato‘Xushu-18'and PFSP‘Xuzishu-3' were used for transcriptome sequencing in this study. By integrated analysis of the published genomic information of sweetpotato and the transcriptome sequencing data of 43 purple-fleshed and 45 non purple-fleshed tuberous roots in public database, the top 50% differentially expressed genes (26 760) with large variation in different samples were identified and selected for WGCNA. A total of 28 gene co-expression modules were identified, in which four modules were specific in purple-fleshed sweetpotato (Grey60 module and Black module are significantly positively correlated to PFSP, while Brown modules and Blue modules are significantly negatively correlated to PFSP). GO analysis showed that the Grey60 module was significantly enriched in the metabolic process of flavonoids and anthocyanin. A total of 47 core hub genes related to anthocyanin biosynthesis were screened in Grey60 module by calculating gene connectivity in the corresponding networks. Of them, MYB113, CHS, three CHI and F3H, GST and LDOX have been previously reported to be related to anthocyanins biosynthesis. The expression patterns of seven of the 47 hub genes were verified by qRT-PCR, which were consistent with the results by transcriptome sequencing. Analysis of the interaction network of hub genes indicated that MYB interacts not only with the known genes related to anthocyanin biosynthesis, such as bHLH, CHI, GST, F3'H and CHS, but also with the transporter genes DUF914 and ABCC4, etc. WRKY3 interacts with many core genes, such as LODX, GST, and CHS. In summary, this study will provide new ideas for the cultivation of high anthocyanin content purple-fleshed sweetpotato varieties and theoretical foundation for dissection of anthocyanin biosynthesis mechanism.
Objective:Licorice is widely used as a traditional Chinese medicine. Glycyrrhizin is the main active ingredient in licorice, a pentacyclic trterpenoid compound, has many pharmaceutical functions, such as anti-inflammatory, antiviral, and liver protection, and has been used in the clinical treatment of COVID-19. With the development of metabolic engineering and synthetic biology, the microbial synthesis of glycyrrhizin and its precursors has been gradually realized. However, the yield is low due to the incompatibility between plant genes and microbial chassis. In last decades, endophytes have been reported to harbor a wealth of functional traits, and could be directly or indirectly used for the production of plant-derived active compounds, indicating that investigation on endophytes has great academic potentials and application values in the field of metabolic engineering. Therefore, this study intends to conduct an in-depth study on the endophyte community of licorice and dig out the microbial-source functional genes that can be used for glycyrrhizin synthesis.Methods:In this study, three year old main root samples of Glycyrrhiza uralensis were collected from Emin County, Tacheng City, Xinjiang Uygur Autonomous Reyion. Metagenomic sequencing of endophytic communities was carried out, and the community structure and functional gene diversity of endophytic communities were analyzed. Through functional gene annotation and phylogenetic analysis, functional genes that may be involved in glycyrrhizin synthesis were excavated.Results:By analyzing the abundance of the community structure, it was found that the dominant endophytes in the licorice root were Steroidobacter denitrificans, Phenylobacterium zucineum, unclassified Phenylobacterium, and Phenylobacterium sp.. The metagenomic data were functionally annotated by COG database, KEGG database, and CAZy database, and the endophytic genes encoding enzymes involved in glycyrrhizin synthetic pathway, such as cytochrome P450 and UDP-glycosyltransferase, were found to be abundant in the endophytes of licorice.Conclusion:This study was the first to use metagenomic sequencing and analysis methods to understand the community structure and functional gene composition of endophytes from G. uralensis. It was proved that the endophytic community contained abundant cytochrome P450 and UGT coding genes, which laid a theoretical foundation for further comprehensive and in-depth study of the biological functions of endophytes from G. uralensis and how they transform into glycyrrhizin biosynthesis resources.
Objective:To screen out the suitable host cells for recombinant goat pox virus (GPV) expressing LacZ gene.Methods:The baby hamster syrian kidney cells (BHK21), sheep kidney cells (SK) and lamb testis cells (LT) commonly used for the culture of GPV were cultured to a single layer in the same cell plate. The monolayer cells were then cultured in the medium containing X-gal, and the blue color of each cell well in the environment of X-gal was observed. RNA was extracted from the monolayer cells and RT-PCR of LacZ gene was performed. PCR products were recovered, ligation vectors were transformed into E. coli (DH5α), and positive clones were selected for sequencing analysis. The β-galactosidase produced by each cell was tested when the monolayer cells were cultured for 72 h.Results:As the culture time prolonged, the gel color of BHK21 and LT wells in solid medium turned blue and deepened gradually. Blue spots were observed under microscope. But SK cells, blank and medium X-gal free did not change color, and no blue spots were observed under microscope. Besides the adherent cells in liquid culture containing X-gal gradually showed detachment, the culture medium was not blue. The electrophoresis band of the RT-PCR product was consistent with the expected molecular weight. The analysis of sequenced results showed that the DNA sequence similarity between the target DNA and the LacZ gene reached 100%, indicating that the 3 cells all have LacZ gene transcripts. The results of β-galactosidase assay showed 3 cells expressed this enzyme, and their production enzyme ability was BHK21>LT>SK, especially SK cells produced very low amount of β-galactosidase.Conclusion:The SK cell screened by the color difference method is suitable for culturing recombinant GPV expressing LacZ gene. The results can provide some reference value for the development of new GPV vaccine.
L-threonine aldolase (L-TA) synthesizes β-hydroxy-α-amino from glycine and aldehyde. β-hydroxy-α-amino acids with two chiral centers is an important pharmaceutical intermediate for the synthesis of many drugs. However, free threonine aldolase is difficult to be reused and separated, which seriously hinders industrial application. Immobilization technology can effectively solve these problems. L-threonine aldolase was immobilized by amino carrier NAA and glutaraldehyde was used as the crosslinking reagent. After optimization of conditions, the optimal immobilization conditions were determined as: enzyme loading 13 U, 0.6 g carrier, glutaraldehyde concentration of 0.4 %(V/V), activation time of 2 h, pH of 8.5 and temperature of 35℃, and immobilization time of 5 h. Under these conditions, the activity recovery of immobilized enzyme was 85.7%. The half-life at 30℃ can reach 59 days, which was 6.5 times that of the free enzyme. The immobilized enzyme was applied in the synthesis of L-syn-3-[4-(methylsulfonyl)phenylserine] (L-syn-MTPS) for 460 h still remained activity of 79.4%. Furthermore, the method to reuse carrier was developed. In this method, the residual amino groups on the surface of the inactivated immobilized enzyme were first activated by glutaraldehyde, and then further combined with the fresh enzyme to realize the reuse of the resin. Using this method, the carrier can be reused twice and the prepared immobilized enzyme can still be used for 460 h. This method greatly reduces the cost of immobilization and lays a solid foundation for the industrial application of immobilized L-TA.
miR-146a is a hot topic in recent miRNA study and keeps highly conserved in different species. It is involved in the occurrence and development of various types of diseases, such as inflammation, autoimmune diseases, cancer and obesity, and its mechanism mainly plays a role through TLR4, MyD88, NF-κB and Akt signaling pathways. In this paper, the roles and mechanisms of miR-146a in different disease processes are reviewed to provide information for further research on the regulatory role of miR-146a in various diseases.
Immune checkpoint inhibitor (immune checkpoint inhibitors,ICIs) activates host anti-tumor immune response by blocking negative regulatory immune signals. Clinical trials have shown that the treatment of ICIs can significantly cause tumor regression in some patients with advanced cancer. In clinical practice, one of the main problems in ICIs treatment is the low response rate. Although various predictive biomarkers such as PD-L1 expression, mismatch repair deficiency, and tumor-infiltrating lymphocyte status have been used to screen patients who respond to treatment, the resistance of ICIs monotherapy still exists. Recent studies have shown that combined anti-VEGF therapy can reduce the resistance of ICIs. VEGF can inhibit angiogenesis necessary for tumor growth and metastasis, while it can reprogram the tumor immune microenvironment and reduce the resistance of ICIs. Many clinical trials have been carried out for the combination therapy of these two targets, and exciting results have been obtained. The mechanism of action of anti-PD-L1 combined with anti-VEGF therapy and the clinical studies of PD-L1/VEGF combined blocking therapy were reviewed and summarized.
Glycosylation can increase the structural diversity of plant natural products, and effectively improve their water solubility, pharmacological activity, and bioavailability, which are critical for the drug development of plant natural products. UDP-glycosyltransferases (UGTs) catalyze the transfer of sugar groups from the activated UDP sugar donors to the acceptors to form glycosidic bonds. The glycosylation of natural products is mainly achieved by UGTs in plants. The rapid growth of plant genome and transcriptome data provides an unprecedented opportunity to explore new UGTs. Three methods have been developed to characterize the catalytic function of UGTs: mutant isolation and cloning, direct gene cloning and characterization, and heterologous probe screening of cDNA libraries. As of December 2020, 412 UGTs have been functionally identified. UGTs belong to the GT-1 family and share a unified conserved sequence (plant secondary product glycosyltransferase, PSPG). The structure of UGTs is mainly solved by the X-ray diffraction technology. The GT-B topology of UGTs contains one N-terminal domain and C-terminal domain both with Rossmann (β/α/β)-like folds. The middle cavity becomes the binding regions of the sugar donors and receptors. MODELLER, I-TASSER and SWISS-MODEL are three commonly used software for building three-dimensional structural models of proteins, which have been widely employed in modeling UGTs structure. UGTs exhibit an inverting catalytic mechanism, usually associated with a bimolecular nucleophilic substitution reaction (SN2), and the highly conserved catalytic dimer (His-Asp) in the active site is essential for the glycosylation activity of UGTs. However, most plant UGTs show low catalytic activity, stability, and substrate specificity, which has limited their industrial application. Recently, the improvement of the catalytic properties of UGTs by molecular modification has achieved significant progress. This review summarizes the following five modification methods for UGTs. The first method, domain replacement combined with site-directed mutation, is mostly used between UGTs with high sequence similarity or between enzymes of the same family to produce new UGTs with different functions. Because they have a highly conservative three-dimensional structure, including the N-terminal domain that recognizes the acceptors and the C-terminal domain that recognizes the UDP-sugar donors, the specificity of the sugar-donor and acceptor substrates is quite different. The key amino acids in or near the catalytic activity pocket usually determine or directly affect the catalytic activity and substrate specificity of UGTs. After multiple sequence alignments, the strategy of replacing non-consensus amino acid residues with consensus sequences at each position is the second method, called activity-based sequence conservation analysis (ASCA), which can improve the substrate specificity and catalytic activity of UGTs. Through three-dimensional structure simulation and protein-ligand interaction analysis, the structural characteristics of active sites and their effects on functions are deeply studied. Rational design based on the structure-function relationship is the third method and has become a powerful means of molecular modification for UGTs. Directed evolution does not require an in-depth understanding of the spatial structure and catalytic mechanism of UGTs. The fourth method is to simulate the natural biological evolution process by utilizing error-prone PCR or semi-rational saturation mutation techniques, and screening mutations with optimized performance. The last one, the structure-guided directed evolution method, such as iterative saturation mutagenesis (ISM) and combined active site saturation test (CAST), bears the advantages of both rational design and directed evolution. Overall, this review delineates the mining strategies, properties, three-dimensional structure, and catalytic mechanism of plant-derived UGTs, and summarizes the strategies of molecular modification for UGTs, including rational design and directed evolution. It provides guidance for the industrialization of plant natural product glycosides by enzymatic synthesis.
Sporopollenin (SP) is a highly cross-linked natural biopolymer composed of polyvinyl alcohol-like unit crosslinked through ester and acetal linkage. SP forms the outer wall of pollen and spore and can resist physical, chemical and biological corrosion. It is the most robust organic compound in nature and is known as the diamond of plant kingdom. Sporopollenin exine capsules (SEC) are abundant in nature. They have good biocompatibility and no immunity. The rich carboxyl, hydroxyl and phenolic groups make SEC easy to be functionalized or complexed with nanomaterials. The plentiful nanochannels on SEC increases their specific surface area, supporting capture of cancer cell and biomolecules. The unique properties of SEC lead to their wide applications in drug delivery carriers, oral vaccine carriers, medical imaging, biosensing, cell growth scaffold, microreactor, micro robot, etc. In this review, the physicochemical properties, preparation methods and functionalization of SP as well as the research progress of SEC are discussed. The application prospect, existing problems and future development direction of SEC are summarized.
Advances in synthetic biology made it possible to engineer microorganisms to serve as “factories” to synthesize substances efficiently, and regulate cell activity by adding chemical inducers. However, the toxicity and irreversibility of chemical inducers limited their applications. Optogenetics, which uses light signals of specific wavelengths to regulate the process of cell life, has the characteristics of specificity, reversibility and high spatial and temporal resolution. In recent years, people have modified photosensitive proteins from different sources and developed various optogenetic elements with different wavelengths for the construction of gene circuits, and thus realized the regulation of bacterial protein synthesis and metabolism. Optogenetics technology builds a real-time signal communication between human and bacteria, making the production process more precise and controllable:(1) Drug delivery through bacteria synthesizes therapeutic factors controlled by light;(2) Improve the catalytic efficiency of the target product by controlling the metabolic pathway;(3) Control the formation of living biomaterials under light induction. With further exploration, optogenetic elements with smaller size, more wavelengths and higher efficiency will be developed to realize multi-input regulation of bacterial life activities.
The coding information used to build proteins exists in highly conserved codon table. In nature, organisms use 20 native amino acids to synthesize proteins of different lengths and orders to perform a variety of biological functions. In recent years, with the rapid development of synthetic biology, it is possible to controllably direct incorporation of non-canonical amino acids in protein synthesis. Non-canonical amino acids with functional side groups could extremely expand the structure and function of proteins, which could also be of benefit in the research of new synthetic biological tools and biological processes. The diversity of side chains serves in many fields, such as protein structure research, functional regulation, constructions of new bio-materials and bio-pharmaceutical industry development. This paper introduces the basic principle of the genetic codon expansion technology, and organizes the efficiency optimization strategies as well as new methods of constructing mutant library. In addition, it also summarizes the cutting-edge progress of the codon expansion technology in the field of bio-medicine. Finally, we summarize the current challenges faced by this technology, such as the limited number of available codons, the limited variety of orthogonal translation systems, and the low efficiency of multiple-incorporation of unnatural amino acids. We hope that these contents could help researchers establish suitable methods for the insertion of unnatural amino acids and promote the further development of this technology.
In the latest revision of biotechnology regulations, the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture revised the original regulations on the import, interstate movement and environmental release of organisms developed by genetic engineering to a greater extent. APHIS names it SECURE rule. This rule adopts a new safety evaluation method, shifting the focus of evaluation from the original production method to the characteristics of the product. In addition, the new rules further simplify the supervision method through procedures such as exemption procedures and supervision status review. However, the SECURE rule also has problems such as insufficient coordination between departmental legislation and imperfect construction of supporting mechanisms, which may bring certain challenges to the implementation of the new rules. As a major country of genetically modified technology, China can critically learn from the latest U.S. legislative practice in the legislation of agricultural biotechnology and its product supervision to provide legal protection for the development of the Chinese biotechnology industry.
