Objective: To prepare the exosomes that display the vesicular stomatitis virus glycoprotein (VSVG) by utilizing genetic engineering technology, which promotes membrane fusion. Meanwhile, the exosomes were further modified with aptamers that target dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptors to prepare functionalized exosomes, which is achieved through DNA hybridization chain reaction mediated by nucleic acid probes. This approach reprograms the targeted recognition process between tumor cells and dendritic cells, ultimately enhancing their interaction. Methods: The paper focuses on mouse mammary tumor cells (4T1) and mouse dendritic cells (DC2.4) as research subjects, which demonstrates through confocal imaging and flow cytometry that VSVG-Exos can specifically bind to 4T1 cells via membrane fusion, and that DC-SIGN aptamers can specifically target DC2.4 cells. Results: Functionalized exosomes can reprogram and modify 4T1 cells to enhance their targeted recognition effect with DC2.4 cells. Conclusion: Functionalized exosomes can effectively deliver molecules with specific functions to the surface of tumor cells and reprogram and modify them, thus improving the ability of immune cells to accurately locate and efficiently attack tumor cells. This provides a new approach and strategy for targeted elimination of tumor cells.
Objective: Epithelial cell adhesion molecule(EpCAM) is one of the specific and effective targets of anti-tumor therapy, because the expression of EpCAM is limited to the basolateral epithelial cells in normal cells and is concealed. However, EpCAM is overexpressed in a variety of epithelial cancers, including bladder cancer, and is in a state of easy binding. Since immunotoxins targeting EpCAM have not been applied to the treatment of bladder cancer, new treatments for bladder cancer will be explored. Methods: The immunotoxin was constructed by linking the single-chain antibody of EpCAM 4D5MOCB with toxin PE38KDEL by flexible peptide GGGGS at the molecular level for design and preparation, and expressed in Escherichia coli BL21 (DE3). The binding effect of the immunotoxin on bladder cancer cells and its inhibitory effect on the growth of bladder cancer cells were studied. Results: The quantity of 4D5MOCB-mediated immunotoxin selectively binding to positive cells is about 169 times that binding to negative cells. The immunotoxin could effectively inhibit the growth of 5637, SW780 and RT4 with an IC50 up to 0.5 pmol/L. At the same time, it could inhibit cell colony formation and cell migration and induce cell apoptosis. The immunotoxin did not bind to EpCAM-negative HeLa cells and had no inhibitory activity. Conclusion: The immunotoxin prepared in this study has good selectivity and can effectively inhibit the proliferation and metastasis of positive cancer cells. The preparation of immunotoxin is simpler than that of antibody-drug conjugates (ADCs) and it is more homogeneous than ADCs. This study also provides an experimental basis for the application of immunotoxin in the treatment of solid tumors.
Objective: Due to the higher seeding density and running density of the intensified fed-batch of ultra-high seeding density (uHSD-IFB) process, the feeding strategy of the traditional low-density culture process often does not provide sufficient nutrients for cell maintenance and product expression of the process, which ultimately leads to low process yield and lower production efficiency. By optimizing the feed medium and feeding strategy, the uHSD-IFB process will be successfully established and the target protein yield will be increased. Methods: A monoclonal antibody-expressing CHO-K1 cell line was studied in this paper, a two-stage dynamic feedback feeding strategy with glucose as the control indicator was designed by metabolic kinetics and stoichiometry analysis, and the nutrient concentrations for key trace elements in the feed medium were screened and optimized in combination with design of experiment (DoE). Results: The optimized process effectively alleviated the contradiction between nutrient depletion and accumulation of metabolic by-products in the uHSD-IFB process and achieved the purpose of cell growth and product synthesis in the ultra-high inoculation density culture process. The cumulative titer of the optimized design of the uHSD-IFB process was increased by 95% and the daily yield was increased by about 97% compared to that before optimization. Conclusion: This proposed feeding strategy can provide help to rapidly establish an enhanced fed-batch culture process of ultra-high seeding density with high cell density and high product expression.
The CRISPR/Cas system (clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) has been developed as a new technique widely used in gene editing in recent years. It is derived from the adaptive immune system of bacteria and archaea. At present, the CRISPR system has been extensively explored, among which Class II CRISPR/Cas9 is used as an effective gene editing tool in microorganisms, plants and animal cells. Compared to typical CRISPR/Cas9, CRISPR/Cas12a, which belongs to Class II technology, has also been investigated and developed and has been receiving increasing attention due to its unique advantages. This review aims to introduce the characteristics of CRISPR/Cas12a, its differences from CRISPR/Cas9 in the biological mechanisms, as well as its application in synthetic biology and medicine so as to provide reference for the further development and application of CRISPR/Cas12a gene editing technology.
Endogenous small molecules play vital roles in the cell lifespan, such as supplying the energy and substrates for biological processes and mediating cellular communication. Previous studies have shown that some endogenous reactive molecules can non-enzymatically modify proteins via spontaneous chemical reactions, thereby regulating protein structure and functions and participating in various cellular processes. Recently, the rapid development of proteomics technology for capturing non-enzymatically modified target proteins contributes to elucidating the function of the modified target protein. This review focuses on reversible and irreversible non-enzymatic covalent modifications (NECMs) produced by active metabolites, respectively, and introduces the formation mechanism of NECMs and the application of proteomics in the discovery of new post-translational modifications (PTMs) and the elucidation of metabolites-regulated protein functions.
The microfluidic chip based on loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method that integrates nucleic acid extraction, LAMP reaction and signal display on a chip of several centimeters. It has advantages such as high sensitivity, strong specificity, convenient and fast operation, and a detection process not easy to be contaminated and can realize the detection process from sample to answer. It has become a hot spot in the current detection field. In this paper, the principle of LAMP reaction, the types of microfluidic chips and the research progress of LAMP microfluidic chips in the detection of pathogens and tumors are reviewed, and the existing problems and future development prospects of LAMP microfluidic chips are analyzed. This review may help further expand the understanding of new detection technologies and accelerate their application in on-site rapid detection.
African swine fever (ASF) is a high-mortality infectious disease in pigs caused by the African swine fever virus (ASFV). ASF spreads in more than 40 countries around the world, and the ASF epidemic occurred in China in 2018, which caused serious economic losses to the pig industry. The World Organization for Animal Health (WOAH) lists it as a notifiable animal disease, and China lists it as a Class A infectious disease. Due to the complex genome structure and immune escape mechanism of ASFV, the development of ASF vaccine is difficult. No safe and effective ASF vaccine is available until now, and epidemic prevention and control mainly relies on accurate detection and strict biosecurity measurements. Therefore, the establishment of rapid, accurate and efficient diagnostic methods is essential for ASF prevention and control. In order to promote the research and development of new diagnostic methods to improve detection efficiency and clinical application, the current research progresses of ASF diagnostic methods are reviewed, focusing on the selection of diagnostic targets, as well as the research and development status, and advantages and disadvantages of etiological and serological detection methods. For example, there is introduction of droplet digital PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system) and other new etiological diagnostic technologies and new serological detection methods that use nanomaterials, screening of dominant epitopes, and multi-antigen epitopes to improve sensitivity and specificity. Overall, this review provides a reference to develop new diagnostic methods for ASF epidemic prevention and control.
Enzymes with high efficiency and specificity have attracted much attention from researchers. Among them, metalloenzymes account for about 1/3 of natural enzymes. Metalloenzymes are generally composed of metal cofactors and corresponding protein scaffolds, in which the metal cofactors provide the active center. The protein scaffolds provide the chiral environment and attachment sites for metal cofactors. Existing studies have revealed that metalloenzymes fail to work without metal cofactors. The metal cofactors mainly exist in the form of metal ions or metal ligands. Among the natural metalloenzymes discovered so far, the metal elements in metal cofactors are mainly Fe, Cu, and Zn. Besides, there are also Mn and other metal elements. Metalloenzymes play an important role in organisms, including signal transduction and immune regulation. Various metalloenzymes can catalyze different reactions, such as hydroxylation and epoxidation. However, it is difficult for natural metalloenzymes to catalyze nonnatural substrates. Some metalloenzymes have low catalytic efficiency and poor stability in vitro, making them unable to be widely used. Recently, rapidly developed biotechnology has accelerated the development of metalloenzymes. By simulating natural metalloenzymes, artificial metalloenzymes (ArMs) have been constructed continuously. The appearance of ArMs has expanded reaction types. In summary, three main strategies have been applied in designing ArMs, including the reconstruction of cofactors, design of protein scaffolds, and modification of nanoparticles. The reconstruction of cofactors is mainly achieved by chemical modification and replacement. Design of protein scaffolds is achieved by selecting some stable structures and utilizing computer-aided methods. Notably, the development of nanotechnology has also provided good ideas for redesigning ArMs. The enzyme property can be improved by binding metalloenzymes to the surface of nanometers or being embedded in nanoparticles. Herein, we summarize some achievements of ArMs in recent years. A brief introduction about the challenges and opportunities faced by ArMs is provided, which is helpful for the design and application of ArMs.
Cyanobacteria are an important group of photoautotrophic and prokaryotic microorganisms, which can directly convert carbon dioxide and solar energy into high value-added chemicals. Due to low nutritional requirements, rapid growth and well-established genetic manipulation tools, cyanobacteria are considered to be a model system. Scytonemin is a lipid-soluble indole alkaloid pigment, which is found in some cyanobacteria under UV-A radiation. It can strongly absorb UV-A radiation and has great pharmacological potential with interesting anti-inflammatory and anti-proliferative activities. Thus it has great prospects in cosmetic and biomedical applications. So far, the mechanism and regulation of scytonemin synthesis have been illustrated in Nostoc punctiforme PCC 73102. However, several bottlenecks in genetic engineering have hindered the efficient and sustainable synthesis of scytonemin. By the development of synthetic biology in vivo and ex vivo engineering, the efficient synthesis of scytonemin is expected to be realized. Here, we summarize the scytonemin on its structure, chemical properties, biosynthetic pathways, regulatory mechanisms, stress physiology and applications. Moreover, the future prospects and directions of scytonemin are also discussed.
Tetrapenoids, mainly carotenoids, are widely used in medical and food applications due to their antioxidant, anti-inflammatory, and anti-cancer properties, but traditional methods of extraction from natural products and chemical synthesis have many limitations and are not conducive to industrial mass production due to high costs or poor product activity and purity. In recent years, microbial synthesis has become a trend for synthesizing high-value products based on gene editing and other technologies. This review introduces the main pathways of carotenoid biosynthesis, reviews the progress of carotenoid synthesis in different types of chassis strains in recent years, and summarizes the main optimization strategies used in current research. It shows the recent progress of carotenoid synthesis by microorganisms and the advantages and disadvantages of the comparison between different types of chassis bacteria by comparing the synthesis strategies and yields of different strains, and points out the development direction to further improve the titer of target products by summarizing the main optimization methods. It provides more references for the subsequent research related to the synthesis of carotenoids by microorganisms.
The indolizidine alkaloid swinsonine (SW), which is produced by fungi, induces severe locoweed diseases in mammals. SW is a potential anti-cancer medication. Different fungi have various swainsonine synthesis pathways. A number of genes regulate the secondary metabolism of SW synthesis pathways. Early research revealed that the sac gene can promote the level of SW produced. Subsequently, P5CR (pyrroline-5-carboxylate reductase), a catalytic enzyme for the synthesis of pipecolic acid, has been found in endophytic fungi of locoweed. Later, the SWN gene cluster is proposed and the enzymes that each gene in the SWN gene cluster encodes for are mainly responsible for catalyzing the reactions that transform pipecolic acid to SW. In this paper, the research on the SW synthetic pathways of Rhizoctonia leguminicola, Metarhizium robertsii, and endophytic fungi of locoweed is reviewed.
Objective: To evaluate the biomedical industry technological innovation capability of major provinces (cities) in China based on high-value patents. Methods: Firstly, based on the standards put forward by Chinese National Intellectual Property Administration, the high-value patents are defined. Secondly, according to the characteristics of high-value patents, the evaluation indicators are selected from three dimensions: industrial technology innovation investment, industrial technology innovation output and industrial technology innovation quality. Finally, the weight is determined by the analytic hierarchy process (AHP) method. Results: Using the established indicator system, the biomedical industry technological innovation capability of Jiangsu, Beijing, Guangdong, Shandong, Zhejiang and Shanghai were evaluated in three stages. It was found that the biomedical industry technological innovation capability of Jiangsu and Guangdong have been constantly improved, while the capability of Shandong and Zhejiang are unstable and the capability of Beijing and Shanghai are declining. Conclusion: Although the overall evaluation of the biomedical industry technological innovation capability cannot be completed entirely by patent information, the evaluation of technological innovation ability of biomedical industry based on high-value patents can provide reference for the development of biomedical industry in China.
