Objective: To investigate the effects of cleavage and polyadenylation specific factor 6 (CPSF6) on the proliferation, migration, invasion and ATP production of human glioblastoma (GBM) cell lines U87 and U251, and to further investigate the related regulatory mechanism. Methods: First, the expression levels of CPSF6 in GBM tissues and paired non-tumor tissues were detected by western blot and immunohistochemistry and analyzed by an online database, and the relationship between CPSF6 and the histological grade of GBM and patient prognosis was also analyzed. CPSF6 was knocked down in U87 and U251 cells with short hairpin RNA (shCPSF6). The expression of CPSF6 in U87 and U251 cells was detected by real-time quantitative PCR and western blot, respectively. After knocking down CPSF6, the proliferation ability of GBM cells was tested by CCK8 assay, and the migration and invasion ability of GBM cells was detected by Transwell assay. ATP assay was performed to detect changes in intracellular ATP levels and to determine the oncogenic role of CPSF6 in GBM. RNA-seq was used to analyze mRNA 3'UTR changes in GBM after CPSF6 knockdown, and KEGG enrichment was used to analyze signal pathways related to different target genes. Under the instructions of the enriched signal pathway, the protein expression levels of LC3 and Beclin-1, which are related markers in the autophagy signaling pathway, were detected by western blot assay after knocking down CPSF6. Transmission electron microscopy was used to observe the occurrence of intracellular autophagy in GBM cells in the experimental and control groups. Results: CPSF6 was significantly up-regulated in GBM tissues compared with the paired non-tumor tissues, and the high expression of CPSF6 was associated with poor prognosis in patients. After CPSF6 was knocked down, the proliferation, migration and invasion of GBM cells were significantly reduced, and the intracellular ATP level was decreased. Bioinformatics analysis, transmission electron microscopy and western blot assay demonstrated that CPSF6 potentially promoted the activation of autophagy pathway. Conclusion: CPSF6 was upregulated in GBM. The high expression of CPSF6 is associated with poor prognosis in patients and is positively correlated with histological grade of GBM. CPSF6 exerts oncogenic effects in GBM, and potentially promotes the proliferation, migration, invasion and ATP production of U87 and U251 cells. Knockdown of CPSF6 potentially activates the autophagy pathway in GBM.
Objective: To express metallothionein HcMT of Halostachys caspica in prokaryotic cells and explore its antioxidant activity, so as to lay a foundation for its application in the field of cosmetics. Methods: the prokaryotic expression vector pET-32a-HcMT was constructed, transformed into Escherichia coli BL21, cultured under Zn2+ stress (the final concentration was 200 μmol/L), separated and purified to obtain Zn-HcMT, and its free radical scavenging activity and total antioxidant capacity were measured. The complex Zn-HcMT/TiO2 was prepared and characterized by FTIR. Results: The fusion protein Zn-HcMT was obtained by prokaryotic expression. It had strong scavenging activity for ·OH,O2·- and DPPH free radicals. The IC50 of ·OH and O2·-were 0.386 mg/mL, 0.038 mg/mL, respectively. The DPPH clearance rate of 0.01 mg/mL fusion protein was (37.43 ± 0.006 8)%, the TEAC value of 0.3mg/mL fusion protein was (1.023 ± 0.01)mmol/L, and the reducing power of 0.3 mg/mL fusion protein A700 was 0.142 ± 0.055. FTIR spectra showed the absorption characteristics of Zn-HcMT and TiO2 at the same time. Conclusion: Zn-HcMT has a good ROS scavenging activity and strong antioxidant capacity. It has great application potential in the field of cosmetics.
Objective: To explore new thermostable D-lyxose isomerases that can efficiently catalyze the synthesis of rare sugars from the metagenomes of high temperature hot springs. Methods: Metagenomic DNA was extracted from the sediment of Jifei Hot Spring in Changning,Yunnan and subjected to high-throughput sequencing. The D-lyxose isomerase genes were identified by gene annotation and sequence alignment,and heterologous expression vectors were constructed and induced in E. coli. Two recombinant D-lyxose isomerases were purified by affinity chromatography and were characterized. Results: Eight D-lyxose isomerase genes were identified from the metagenomic sequencing results of the Jifei Hot Spring sediment. Four genes were selected for heterologous expression,among which JF-LI1 and JF-LI4 were successfully expressed in E. coli and their enzymatic activities were detected. The optimum temperatures of recombinant JF-LI1 and JF-LI4 were 70℃ and 75℃,respectively. JF-LI4 has a wide action temperature and high thermal stability,which retained more than 40% of its enzymatic activity in the temperature range of 30℃ to 100℃. The optimum pH of recombinant JF-LI1 and JF-LI4 were 7.0 and 7.5,respectively,and they have high activity and stability under neutral and slightly acidic conditions. Recombinant JF-LI1 and JF-LI4 have a broad substrate spectrum and are active on L-ribose,L-ribulose,D-fructose and D-mannose in addition to their most active effect on D-lyxose. The catalytic efficiencies of recombinant JF-LI1 and JF-LI4 for L-ribose were 0.56 and 0.61 L/(mmol·s-1),respectively,which were the highest among the known D-lyxose isomerases. Conclusion: Eight new D-lyxose isomerase genes were obtained from the high temperature hot spring metagenome,and two of them were heterologously expressed and characterized. They have high pH stability,strong thermal stability and wide substrate specificity,which enable them to have important application potential in pharmaceutical industry, food, cosmetics and other industrial fields.
Objective: GM1 gangliosidosis is a devastating lysosomal storage disease featured by progressive and fatal neurodegeneration, caused by a mutation in the galactosidase beta 1 (GLB1) gene that reduces the activity of human beta-galactosidase. Currently, no effective therapy exists for the affected individuals, and AAV gene therapy is viewed as the most promising method. The object of this research is to obtain a mutant of GLB1 with higher β-gal activity through genetic modification, so as to be used in subsequent AAV-mediated gene therapy. Methods: The sequence of galactosidase beta 1 gene from human and other six vertebrates was analyzed by multiple sequence alignment, and some amino acid sites were selected for modification. HEK-293 cells were transfected or infected with either the recombinant plasmid or rAAV9 carrying the mutant site, and the activity of the mutant was compared with that of the control. The rAAV9 virus carrying coGLB1-R299L was injected into GM1 mice to explore the expression of the mutant in vivo. Results: The coGLB1-R299L mutant was screened from 15 mutants and then transfected into HEK-293 cells. It displayed higher β-gal activity with a 30%~40% increase compared with that of the wild-type amino acid sequence. The β-gal activity of rAAV9-coGLB1-R299L group was 2.2 times higher than that of the cell control group in the AAV infection experiment in vitro. The results in vivo showed that rAAV9-coGLB1-R299L was widely expressed in the GM1 mice, and the β-gal enzyme activities in the heart, liver, spleen, lung and brain tissue were significantly increased. Conclusion: A mutant coGLB1-R299L with higher β-gal activity is obtained, and the in vitro expression and distribution of enzyme activity of rAAV9-coGLB1-R299L in model mice was preliminarily explored, laying a foundation for the application of this mutant in the treatment of AAV-mediated GM1 gangliosidosis.
The development and application of molecular point-of-care (POCT) technology has been noticed in recent years because of its high sensitivity, fast analysis speed, small size, and low detection cost. The development and application of molecular POCT technology is of great significance for protecting human life and health and coping with the threat of emerging and sudden infectious diseases. In this paper, several emerging molecular POCT technologies in the past five years, focusing on describing the latest research progress, and their application prospects were introduced. The advantages and challenges of molecular POCT were discussed, as well as the technical strategies to improve its detection sensitivity and selectivity.
With the outbreak of COVID-19, the world urgently needs a large number of effective vaccines to deal with this disaster. mRNA vaccines are safe and have short development cycle, which can fill the gap between epidemic diseases and vaccine shortages. So mRNA has become one of the most potential vaccines at present and has attracted attention in the field of infectious diseases and tumors. Technological innovation has greatly improved the shortcomings of mRNA, such as instability and low translation efficiency. However, delivering mRNA to target cells safely and efficiently is still a major challenge that hinders the progress in mRNA research. Hopefully, delivery systems have put forward many effective solutions. This review focuses on the non-viral vector delivery system for mRNA vaccine delivery in vivo, and the application of mRNA in infectious disease and tumor vaccine, in order to provide reference for research and development of mRNA vaccines.
Ongoing efforts in synthetic biology are focused on the design of reusable and modular fragments that demonstrate well-characterized behaviors and functionalities in biological systems. In the cell-free protein expression system, riboregulators are developed as an important sensing element in point-of-care testing, which can change their structure through the induction of target molecules, thereby regulating the expression of downstream genes. Different types of riboregulators and their action mechanisms are systematically introduced, including traditional riboregulators, toehold switch, toehold repressor, three-way junction repressor, small transcription activating RNA, and riboswitch. The main processes of constructing riboregulators are described in detail: designing with computers, testing gene expression, and analyzing the functional structure. Finally, the applications of in vitro point-of-care testing (POCT) based on riboregulators are summarized, mainly including pathogen nucleic acid detection based on toehold switch and small molecule detection mediated by riboswitch. The characteristics, challenges, and development tendencies of POCT in the cell-free system are discussed and summarized. In conclusion, the construction and application of riboregulators have opened up a new direction in the biosensing field. This article is expected to provide some helpful insight into the development of new riboregulators and POCT tools.
The genome editing system can bring the precise modification of DNA or RNA, which provides great help for the development of life sciences. The CRISPR/Cas9 could induce double or single strand DNA damage at target sites. The damages are repaired by non-homologous end joining (NHEJ) without donor template or homologous recombination (HR) with donor template in cells. The HR-based genome editing strategy is common to generate precise modification of DNA. However, NHEJ plays a dominant role in mammal DNA repair. To improve the HR efficiency,the researchers designed multiple strategies, which include genome editing system optimization and DNA repair pathway regulation. The related research achievements from the aspects of DNA damage repair pathway, Cas9 mutation selection, sgRNA design, donor template design, functional regulation of DNA repair pathway related proteins, improvement of donor template recruitment efficiency, cell cycle regulation and improvement of editing cell survival efficiency were reviewed. A one-size-fits-all HR promotion strategy has not yet been developed. The case-specific strategies of HR-based are required during relative research. This review can provide theoretical reference for improving the efficiency of CRISPR/Cas9-mediated HR in animal genome editing and provide help for animal gene function analysis, gene therapy and economic animal breeding through gene editing.
Polyphenols, a typical class of natural products in our daily diet, have functions of interfering with molecular signaling pathways, affecting the composition of gut microbiota, and protecting human health. Biofilm formation and microbial pathogenicity can be interfered by various polyphenols targeting different microbial quorum sensing (QS) systems, which provides a theoretical basis for the clinical applications of polyphenols. However, the complexity of polyphenol-based interference involves multifarious compounds, various microorganisms and intricate microbial phenotypes. First, a brief summary of types, sources, and amounts of polyphenols is introduced, including flavonoids, phenolic acids, stilbene, lignans, and others based on the number of phenolic rings and structural elements. Then the polyphenol-based interference targeting diverse QS-relevant phenotypes is summarized, including microbial motility, microbial adhesion, biofilm formation, and the release of virulence factors. Furthermore, various interference effects of some common polyphenols on the typical pathogens are summarized to contribute to potential applications. Finally, some key challenges and general perspectives on polyphenol-based interference for various future therapeutic applications are identified.
Industrialization has led to various toxic compounds emission, which causes environmental contamination. Aside from natural geological weathering, traditional solutions include chemical conversion, physical absorption, electrochemical methods and ions exchange, but their applications are limited due to some disadvantages, such as secondary pollution, high energy requirement, high investment cost, low regeneration efficiency and inefficiency in low-concentration wastewater treatment. Cell surface engineering is an innovative, cost-effective biotechnology of microorganism for modification of cell surface function through joining external functional peptides with surface anchoring proteins. In contrast to conventional intracellular and secretion expression systems, proteins displayed on the surface of microorganism may exhibit enhanced stability against changes in organic solvents, proteases, temperature and pH. Surface-engineered cells prepared by cultivation are ready to be used as microparticles covered with proteins/peptides, avoiding troublesome concentration procedures and protein purification. Furthermore, cell-surface display engineering is suitable for high throughput screening from the mutant library for more capable proteins/peptides at the single-cell level. Currently, this technology is widely used in the control of environmental pollution. This review is focused on recent strategies of using cell-surface display technology in environmental bioremediation, summarizing its applications, recent progress and future prospects.
During the 13th Five Year Plan period, the Ministry of science and technology set up the National Key Research and Development Program ‘stem cell and transformation research’. After five years of implementation, the program has made important progress. Through reviewing the approval and implementation of the project, this paper summarizes the experience and shortcomings in the management, and puts forward relevant suggestions for stem cell research and deployment in the 14th Five Year Plan, so as to further enhance the core competitiveness of China’s stem cell and transformation research, and accelerate the promotion of stem cell research results to benefit people’s health.
Biosensor is immobilized biosensitive material as identification element, which has the characteristics of small size, high specificity, fast response and high accuracy, possessing a broad market application prospect. In recent years, biosensor technology has developed rapidly, and research achievements have emerged continuously. In order to better understand the research status of China’s biosensor industry and further promote its healthy development, based on the IncoPat patent database, ITGinsight is used as a literature analysis tool. The overall research situation, regional layout, technical composition and research hotspots in the field of internal biosensors are analyzed, and the core patents are identified. The results show that although China ranks first in the world in the number of biosensor patents, there are problems such as unbalanced regional distribution of scientific research strength, weak research and development strength of enterprises, and inadequate mastery of key core technologies. Finally, corresponding countermeasures are put forward according to the shortcomings of biosensor research in China, which can provide reference for the rapid development strategy of biosensor technology in China.
