Objective: To investigate whether General transcription factor II subunit 2(GTF2H2) affects the proliferation and migration of Hep3B cells and the underlying molecular mechanism. Methods: The GTF2H2 knockdown Hep3B cell model was constructed by transfecting GTF2H2-siRNA. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the GTF2H2 knockdown effect in Hep3B cells. MTS cell proliferation assay kit was used to detect the proliferation ability of GTF2H2 knockdown Hep3B cells. The migration ability of GTF2H2 knockdown Hep3B cells was evaluated by cell Transwell assay. Western blotting was used to detect whether GTF2H2 knockdown affects tumor associated molecular signaling pathway. Results: The proliferation and migration ability of GTF2H2 knockdown Hep3B cells was stronger than that of the controls. Western blotting showed that the expression of p-AKT pathway protein in GTF2H2 knockdown Hep3B cells was significantly increased. Conclusion: GTF2H2 may affect the proliferation and migration ability of Hep3B cells by the regulation of the AKT molecular signal pathway.
Protein homeostasis through protein quality control is critical for bacterial growth and adaptation to host immune stress. The Clp protease plays an important role in protein degradation and homeostasis in Mycobacterium tuberculosis. There are two proteins in the Clp protease that are responsible for recognizing unfolding substrate proteins:ClpC and ClpX. Then unfoled substract is transferred into the chamber of ClpP, where proteolysis is carried out by protease. In order to explore the respective functional characteristics of ClpC and ClpX in mycobacterium,Mycobacterium smegmatis was selected as the experimental strain, and knockdown strains of ClpC and ClpX were constructed successfully by the CRISPRi method. The results showed that the ClpC and ClpX knockdown strains showed significant growth phenotypes differences compared with wild Mycobacterium smegmatis. Low expression of both ClpC and ClpX severely affected the growth of Mycobacterium smegmatis. ClpC low expression resulted in the loss of biofilm formation ability of the strain, while that of ClpX caused incomplete cell wall and bacterial filamentation indicating that ClpC and ClpX may have different physiological functions in mycobacterium.
Objective: To establish a recombinant adenovirus vector construction method based on the DNA Assembly method. Methods: First, by designing suitable restriction sites and homology arms, the backbone plasmid pChAd63 of Chimpanzee adenovirus serotype 63 (ChAd63) was obtained using traditional restriction endonuclease ligation methods and the DNA Assembly method. Subsequently, the shuttle plasmid pShuttle63/EGFP carrying the EGFP gene was digested with Sca I, and pChAd63 was digested with Hpa I. The recombinant adenovirus vector pChAd63/EGFP was obtained by the DNA Assembly method. Finally, the recombinant adenovirus rChAd63/EGFP was rescued in 293 cells. Results: The recombinant adenovirus pChAd63/EGFP was successfully constructed by the DNA Assembly method, and the recombinant adenovirus rChAd63/EGFP was rescued. Conclusion: The DNA Assembly method can be widely used in the construction of recombinant adenovirus vectors, and is beneficial to improveing the construction efficiency of recombinant adenovirus vectors.
Objective: In order to improve the on-target effect of CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9).At the same time, it can prove the application value of the yeast hybridization system in the study of off-target effect. Methods: Based on the activase gene-edited rice strains previously constructed in our laboratory, the T7 endonuclease I assay was firstly used to predict off-target rates in 30 gene-edited rice strains. Subsequently, the yeast hybrid system was further used to investigate the off-target rate. During this period, at first, standard sgRNA and truncated sgRNA of on-target gene were cloned into the CRISPR/Cas9 expression vector pDW3769 to be recombinant vectors pHZ2 and pHZ4, and then those vectors were transfered into YPH499 yeast haploids to be the recombinant yeast YpHZ2 and YpHZ4. Seven off-target sequences A, B, C, D, E, F, G derived from off-target site prediction and on-target sequence were selected and cloned into the high copy vector pDW3133 and the low copy vector pDW3134, both of which contained the reporter genemCherry. The corresponding high copy recombination vectors were pHZ5, pHZ7, pHZ9, pHZ11, pHZ13, pHZ15, pHZ17 and pHZ19 and the corresponding low copy recombination vectors were pHZ6, pHZ8, pHZ10, pHZ12, pHZ14, pHZ16, pHZ18 and pHZ20, and all of them were respectively transformed into YPH500 yeast haploids to be recombinant yeast YpHZ5-20. Subsequently, recombinant yeast YpHZ2 and YpHZ4 individually hybridized with recombinant yeast YpHZ5-20, colonies of diploid yeast were picked and the fluorescence values were examined at various time periods, and at last off-target rate on the basis of fluorescence values was predicted. Results: The significant and stable fluorescence value was at 144-192 h, and off-target sgRNA sequences which have higher homology to the targeted sgRNA became more likely to be edited by CRIPSR/Cas9, too. However, truncated sgRNA can reduce the off-target rates compared with standard sgRNA. The result of off-target detection according to rice plants showed about 20%, and the off-target detection based on yeast hybridization was about 20%-28%. Conclusions: The fluorescence values are most significant and stable during the stationary phase in batch culture of yeast and directly proportional to the copies of the vector, and sgRNA sequences and structure can affect and targeting of CRISPR/Cas9. The prediction results of off-target rate were similar between the two methods so that the yeast hybrid platform can be proved to be a highly useful method in evaluating the off-target rate of the CRISPR/Cas9 system and investigating factors affecting the off-target rate.
The rapid development of the biopharmaceutical industry, especially the increase in upstream expression and the expansion of scale, had promoted the perfusion culture for upstream. This also promoted the continuous purification strategy for the downstream purification process. In order to compare the purity and yield between batch purification and continuous purification, taking the perfusion of Fc fusion protein as an example, the experiment was carried out with BioSMB PD equipment, and on this basis, process scale-up and production cost evaluation were carried out. The continuous purification realized the continuity of perfusion culture and affinity chromatography. Compared with batch purification, the continuous purification process has many advantages. It can observably reduce the amount of the chromatography resin, improve the efficiency of packing, and reduce buffer used. The production efficiency is significantly improved. The production costs are significantly reduced.
The establishment of rapid diagnostic methods for pathogens is of great significance for the prevention and control of animal epidemics and public health safety in China. Recombinase-aided amplification (RAA) is a novel isothermal amplification technology in which rapid amplification of DNA or RNA can be carried out at low temperatures, and the entire RAA reaction is simple, rapid, accurate, power-saving and convenient. There are three core substances of this technology, including recombinase, single-stranded DNA binding protein and DNA polymerase, which can replace the traditional PCR thermal cycle chain decomposition process. Therefore, the RAA technology will play a more and more important role in the detection of pathogenic microorganisms. The research advances in the detection of pathogens by the RAA technology was reviewed, which may provide reference for further relevant studies.
The efficiency of extracellular electron transfer between microbial cells and electrodes is a key factor limiting the development of microbial electrochemical technology, and the development of molecular biology has brought bright prospects for improving the efficiency of extracellular electron transfer. The results of engineering four representative electroactive microorganisms (Shewanella oneidensis MR-1, Pseudomonas aeruginosa, Geobacter sulfurreducens and engineered Escherichia coli) in pure culture and mixed culture by means of molecular biology are reviewed. How molecular biology methods adopt corresponding improvement strategies for different electroactive microorganisms are explained, and future research directions are prospected.
Terpenes are a large class of highly diverse natural products with various physiological activities such as anti-tumor, anti-oxidation and immune regulation. Therefore, they are widely used in the fields of medicine and health, food, cosmetics, and biofuels. However, obtaining terpene compounds directly from natural resources is low efficient, costly, and often has an adverse impact on the ecological environment, making it impossible to achieve green and sustainable production. Microbial synthesis of terpenes has attracted much attention in recent years. Researchers have conducted explorations from the construction and regulation of synthetic pathways, protein engineering, and fermentation process optimization, and have obtained fruitful results. Among them, the efficiency of key enzymes in the synthetic pathway plays an important role in the microbial production of terpenes. Research on key enzymes is of great significance for improving the ability of microorganisms to synthesize terpenes and thus accelerating the large-scale application of microbial production of such natural products. Here, four key enzymes in the synthetic pathway of terpenes including 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), isoprenyl diphosphate synthase (IDS), and terpene synthase (TPS) were introduced. The regulation of catalytic activities of key enzymes via metabolic engineering, protein engineering and synthetic biology to improve the efficiency of microbial synthesis of terpenes as well as the prospects of using microorganisms to synthesize terpenes were also reviewed.
Ginseng, as a traditional Chinese medicine, has high medicinal value. Its main active component is ginsenoside. Ginsenoside is a series of glycosylated triterpenoids, which is also an active compound with health care function. It is stored in the roots, stems, leaves and flowers of Acanthopanax plants. The content and composition of ginsenoside vary in different kinds of ginseng and different parts of a plant same as. Ginsenoside has a good therapeutic effect on metabolic syndrome and cardiovascular disease.Compared with ginsenoside, rare ginsenoside has better biological activity and is more conducive to body absorption.However, the structure of rare ginsenosides is complex and its content in ginseng is very low, which limits its development and utilization. With the development of biotechnology, the use of biological synthesis of rare ginsenosides has become a research hot spot in this field.Therefore, the research on the synthesis of rare ginsenosides by biological methods in recent years is summarized, and the main types and structures of rare ginsenosides and the research progress of the synthesis of rare ginsenosides by biotransformation and heterologous synthesis are summarized in recent years. The biotransformation method summarizes the transformed organisms using ginsenosides Rb1 and Rg1 as substrates, and the heterologous synthesis method summarizes the biosynthetic pathway of ginsenosides and the enzymes that form ginsenosides with diverse structures. The problems in the biosynthesis of rare ginsenosides were discussed, and its prospects and future research directions were prospected, in order to provide more biological clues and preparation strategies for ginseng researchers.
Biomedical industry is the third largest industry in China’s pharmaceutical manufacturing industry and also one of the emerging high-tech industries supported by Guangdong-Hong Kong-Macao Greater Bay Area. It is necessary to further guide the construction planning of biomedical industry in Guangdong-Hong Kong-Macao Greater Bay Area by analyzing the relationship between the main business income and various indicators. The grey comprehensive correlation analysis method is an important tool to explore the correlation degree between indicators. Through various authoritative channels, six index data are obtained, including the number of enterprises above designated size, business incubators, new patents, projects approved by non NSFC, industry university research cooperation projects and the total investment of projects approved by NSFC. The grey relational analysis method is used to model and solve the data and the correlation degree between the six indicators and the main business income of Guangdong-Hong Kong-Macao Greater Bay Area biomedical industry is obtained. The results show that the number of enterprises above designated size has the highest correlation with the main business income of the biomedical industry in Guangdong-Hong Kong-Macao Greater Bay Area, followed by the total investment of NSFC funded projects and the number of industry university research cooperation projects. The number of non NSFC funded projects ranked fourth, the number of business incubators and the number of new patents ranked fifth and sixth, respectively. Therefore, in order to further develop the biomedical industry in Guangdong-Hong Kong-Macao Greater Bay Area, three aspects should be focused on, increasing the number of enterprises above designated size, attaching importance to the investment of NSFC funded projects and strengthening the cooperation of industry.
The commercial planting of genetically modified (GM) insect-resistant (IR) maize has become one of the main thrusters for the substantial increase in agricultural productivity. In recent years, the scale of industrialization of GM IR maize has been continuously expanded, effectively controlling the occurrence of target pests, reducing the application of chemical pesticides, and providing an important guarantee for food security and increasing farmers’ income. The global development status of GM IR maize was introduced, and the ecological problems related to the cultivation of GM IR maize was also analyzed. At last, some suggestions were raised for promoting the industrialization of GM IR maize in China.
Antibody-drug conjugates are immunoconjugates comprised of monoclonal antibodies tethered to a cytotoxic drug via a chemical linker.It has been proven to be a promising technical method in the clinical treatment of hematologic malignancies and solid tumors.Therefore,it has newly become one of the fastest growing areas in the researching and developing of anticancer drugs at home and overseas. Five key requirements of ADCs throughout the research and pharmacuetical manufacturing process were expatiated systematically,and the pipelines and strategic layout of major domestic companies were also analyzed in order to provide a reference for the R&D layout of ADCs for the emerging companies and industrial decision for regional government.
The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses tremendous threats to public health and urgently needs an effective cure. The progress in drug repurposing, therapeutic antibody and small molecule development, and traditional Chinese medicine for the treatment of COVID-19 in the early state of the outbreak were focused. A number of drugs have been revealed to inhibit the replication of SARS-CoV-2 by drug repurposing, which includes remdesivir, favipiravir, chloroquine, and hydroxychloroquine. Remedsivir is the first drug approved by the FDA for the treatment of COVID-19 in USA. As for the development of innovative drug, a major effort has been directed to the discovery of therapeutic antibodies targeting spike protein and inhibitors of 3C-like protease as well as RNA-dependent RNA polymerase of SARS-CoV-2. In addition, traditional Chinese medicine has played an important role in the prevention and treatment of COVID-19. Jinhua Qinggan granule, Lianhua Qingwen capsule, Xuebijing injection, Shuanghuanglian oral liquid, Qingfei Baidu decoction, Huashi Baidu prescription, and Xuanfei Baidu prescription have entered clinical trials for the treatment of COVID-19.
The global pandemic of COVID-19 has taken a major impact on global public health, and social and economic operations. In the case of delays in drug development and insufficient verification of vaccine effectiveness, the priority is to take a large-scale rapid screening to filter out potential infections (especially mild and asymptomatic patients), isolate those infected patients centralized to cut off transmission routes and to protect the susceptible people. Therefore, early diagnosis of SARS-CoV-2 infection is particularly important. This paper summarizes the rapid detection products toward SARS-CoV-2 antigens in the current market, analyzes the global market of rapid SARS-CoV-2 antigen detection, outlines its research and development trends, and in the end discusses the prospects for developing the capability of independent innovation in new technologies and methods for SARS-CoV-2 antigen detection in our country.
With the continuous spread of the COVID-19 epidemic, it is urgent to develop effective therapeutic drugs. Neutralizing antibodies, as the most promising specific therapeutics against SARS-CoV-2, are proved to be effective in clinical trials. The research progress of neutralizing antibodies were summarized, including the involved technologies, and the clinical results in order to provide benefits for developing neutralizing antibodies in emerging infectious diseases including COVID-19.
