Objective:To explore the role and mechanism of miR-17-5p in the autophagy pathway mediated by Mycobacterium tuberculosis by studying the regulatory mechanism of miR-17-5p on autophagy-related gene ATG7 and its effect on cell autophagy. Methods: The target gene ATG7 of miR-17-5p was obtained by bioinformatics analysis. The wild-type(pMirGLO-ATG7-3'UTR-WT) and mutant vector of ATG7 were successfully constructed. The targeting relationship between miR-17-5p and ATG7 was verified by double luciferase reporting system and Western blot. THP-1-derived macrophages infected by Mycobacterium tuberculosis (H37Ra) were divided into three groups: miR-17-5p mimics, miR-17-5p inhibitors, and miR-17-5p nc. The effect of H37Ra infection on the expression of miR-17-5p was detected by quantitative real-time PCR (qRT-PCR). The expression of LC3 protein and the number of autophagosomes were detected by Western blot and immunofluorescence. Results: MTB infection can cause miR-17-5p down-regulation, with the increase of infection plural decreased significantly. Bioinformatics predictions showed that miR-17-5p and ATG7 were targeted. Dual luciferase reporter assay and Western blot confirmed that miR-17-5p could bind to ATG7 and negatively regulate it. Western blot and immunofluorescence assay showed that the expression of LC3 II was down-regulated and the expression of autophagosomes was down-regulated in the miR-17-5p mimics group, but the reverse was found in the miR-17-5p inhibitor group. The expression of ATG7 and LC3 II protein in H37Ra infected group was higher than that in uninfected group. Conclusion: miR-17-5p directly targets ATG7 3'UTR to inhibit autophagy and plays a role in the anti-MTB effect of macrophages.
Epoxide hydrolases can catalyze the kinetic resolution of racemic epoxides,retaining enantiopure single enantiomers of epoxides. The catalytic properties of Phaseolus vulgaris epoxide hydrolase (PvEH1) towards phenyl glycidyl ether and its methyl derivates were assayed.Seven residues of PvEH1 were then selected for site-directed mutagenesis based on the results of molecular docking simulation and multiple sequence alignment,followed by single-site and combinatorial mutagenesis to obtain mutants possessing enhanced catalytic properties towards ortho-methylphenyl glycidyl ether (1a). The substrate spectrum analysis showed that PvEH1 displayed both the highest activity (157.2U/g wet cell) and enantioselectivity (E=5.6)towards 1a.Thus, 1a was selected as the model substrate.Among the constructed seven E.coli transformants expressing single-site mutant of PvEH1,E.coli/pveh1 L105I and E. coli/pveh1 V106I exhibited notably improved EH activity and E value.Compared with E.coli/pveh1, the EH activity and E value of E.coli/pveh1 L105I/V106I were improved by 2.1 times and 50%, respectively.Additionally,the specific activity (17.6U/mg) and the catalytic efficiency [17.7L/(mmol·s)]of purified PvEH1 L105I/V106I were 1.5-and 2.1-fold those of PvEH1. SDS-PAGE analysis indicated that the soluble expression level of target protein was enhanced by the combinatorial mutagenesis.The kinetic resolution of 100mmol/L 1a by E.coli/pveh1 L105I/V106I whole cells afforded (R)-1a (ee>96%)with 31.2% yield and a space-time yield of 5.12g/(L·h). Therefore, the superior enzymatic properties will make E.coli/pveh1 L105I/V106I a promising biocatalyst for the preparation of optically pure (R)-1a.
Objective: A new material with ultra-large surface area named nano-forest is prepared by micro-electro-mechanical system(MEMS) processing technology. Based on this material, a new microfluidic chip for point-of-care test with simple operation, rapid detection and high sensitivity is created. Methods: The fabrication of nano-forests in micro-channel on quartz substrate mainly includes, cleaning and drying of quartz substrate; spinning polyimide(PI) coating; re-spinning phenolic resin photoresist on PI coating; photolithography to expose the channel; treating the PI layer with oxygen plasma and argon plasma to synthesize nano-fiber forests structure; nano-fiber-quartz nanoforests are realized by using nano-fiber forests as nanomasks in anisotropic etching of quartz by using reactive ion etching (RIE); the micro-channel with nano-forests structure inside is achieved after removing upper nanofiber forests structure and phenolic resin photoresist coating.The height, width, density and specific surface area of nano-forest are studied and analyzed by scanning electron microscope(SEM). Optical properties are tested by ultraviolet-visible spectrophotometer. The driving force is characterized by the flow rate of PBS solution.The sensitization effect is evaluated by saturated fluorescence test through antibody and AbFluor 680 dye-labeled secondary antibody. The sample pad, bond pad, micro-channel with nano-forests structure, nitrocellulose membrane and absorbent material are assembled on PMMA substrate in sequence, which is the microfluidic chip. The chip based on the sandwich format with a polyclonal antibody and a AbFluor 680 dye-labeled secondary antibody is used to detect ricin toxin(RT). Results: The scanning electron microscope shows that the nanofiber forests structure is formed on quartz substrate after oxygen plasma and argon plasma bombardment. The single nanofiber is upright on the substrate with a diameter of about 50-100nm, a height of 1.8μm and a density of about 20/μm 2. The quartz nano-forests structure can be obtained after RIE with nano-fibre forests structure as mask and resist removal. The single structure is shaped like a cone. The diameter of the cone bottom is about 100-200nm, the height is about 1.0μm, the density is about 10/μm 2, and the surface area to bottom area is more than 5∶1. Self-driven test provides information of the flow rate of PBS is to be about 5mm/s in the micro-channel on the basis of nano-forests structure. The transmittance of the channel is 89.5% at 680nm wavelength. It shows that the channel has good transmittance, which makes the loss of excitation light or emission light much less, and is conducive to the sensor capturing more signals. With same surface modification, the planar quartz structure has shortcomings of short lasting effect time and low saturation fluorescence intensity. To the contrary, nano-forests structure with ultra-large surface area has a good sensitization effect in the test. RT can be detected sensitively based on the significantly fluorescent intensity.The linear range of detection is from 10pg/ml to 6 250pg/ml and the limit of detection (LOD) is lower than 10pg/ml. Conclusion: The nano-forests structure with good optical properties reduces the requirements of sensor and also makes the choice of fluorescent dyes wider.The three-dimensional structure of the nano-forest has an ultra-large surface area, which increases the amount of antibody compared to the planar structure, and thus improves the sensitivity of detection greatly. Compared with the immunochromatographic test strip, the microfluidic chip has an advantage of high sensitivity, thus the quantitative analysis can be realized within a certain range. Most microfluidic chips require complex equipments to provide driving force, which will make them costly and bulky. Driven by the capillary force, the chip with nano-forests structure inside makes the detection simple and fast. Combined with the miniaturized detection terminal, the platform can be miniaturized, portable, and automated, achieving the goal of simple, fast and efficient analysis. These characteristics make the chip an ideal candidate for the development of rapid detection methods.
Objective: According to GC-FID method, the content and stability of β-propiolactone which was used as an inactivator in the preparation of rabies vaccines were systematically studied. Methods: Gas chromatography condition: Agilent DB-624 (30m×0.530mm×3.00μm)capillary column was adopted. The temperature program: Initial temperature of 80℃ was maintained for 1min, then was raised to 200℃ at the rate of 20℃/min and was maintained for 3min. Column flow: 3ml/min. The temperatures of detector and inlet were 250℃ and 150℃ respectively. The linear velocity of the carrier gas (nitrogen) was set to 25cm/s. The injection volume was 1μl and the split ratio was 2∶1. The sampling mode: Manual injection. Results: BPL showed good linear relationship in the range of 1∶100-1∶32 000 with a correlation coefficient more than 0.999. A recovery study of BPL at three different concentration (1∶200, 1∶1 000, 1∶8 000) of spiked samples with range from 95.04% to 116.86% with RSD less than 3.2% further demonstrated the reliability and feasibility of this method. Moreover, the LOD of established method can be achieved as low as 0.112μg/ml. Conclusion The method is simple, specific, stable and can be carried out at room temperature with a satisfactory result, which reduces the requirements of complicated operation. In a word, the proposed method can meet the need that determines BPL in the process of inactivating rabies virus.
In the process of expressing porcine interferon-α (pIFN-α) by methylotrophic Pichia pastoris under high cells density, methanol concentration is generally controlled at relatively high levels of 5-10g/L with the dissolved oxygen concentration (DO) naturally declining to near 0% level. Heterologous proteins induction by P. pastoris is an extremely high aerobic system. If P. pastoris is subject to the environments of high methanol concentration and low DO for a long period, the cells metabolic activity and heterologous protein expression efficiency of P. pastoris would significantly deteriorate; intracellular methanol could severely accumulate, and the intracellular methanol can not be effectively oxidized by/energizing for methanol dissimilation route to synthesize the targeted protein. A novel periodic methanol induction control strategy was proposed and implemented at 20℃. During the induction period, methanol was firstly controlled at the high concentration level of 8-10g/L with DO level naturally staying at about 0% for 7h. Then raising/maintaining DO around 20% by reducing methanol feeding rate. By doing so, methanol concentration naturally reduced to low level of 0-1g/L, and then the environment of high DO/low methanol concentration was created and kept for another 4h. A total of 6 such cycles was repeated. By periodically shifting methanol concentration and DO in between “high/low” and “low/high” environments, the metabolic activity of P. pastoris could be recovered and maintained at higher levels for longer time; intracellular methanol concentration could be reduced to a very low level (≤ 0.003g/g DCW) but without deteriorating pIFN-α induction, the methanol toxicity was greatly relieved. As a result, pIFN-α activity reached the highest level of 3.90×10 7IU/ml, which was 86% higher than that using the traditional constant methanol concentration control strategy. The proposed periodic methanol induction control strategy showed its effectiveness in pIFN-α production and would supply the useful information/reference for other heterologous proteins fermentation processes.
Immobilized enzymes are biocatalysts that typically have higher functional efficiency and reproducibility than free enzymes. However, the use of enzyme immobilization is still limited, and the research is mainly conducted within model enzymes. In recent years, the study of metal organic frameworks (MOFs) as enzyme immobilization carriers has attracted great attention, and the nature of MOF-enzyme complexes remains to be elucidated. In the present study, a novel immobilized biocatalyst was prepared by embedding a 5-hydroxymethylfurfural oxidase (HMFO) in zeolite imidazole ester skeleton ZIF-8(a typical MOF) through biomimetic mineralization. The morphology of the composite catalyst under scanning electron microscope was different from the classical rhombohedral dodecahedron of MOF immobilized enzyme. Protein concentration assay conducted by Commassie brilliant blue G-250 method indicated that the immobilization efficiency was reached 89.0%. Moreover, the conversion efficiency of HMFO@ZIF-8 to 5-hydroxymethylfurfural reached 84.3%, and the yield and selectivity were significantly improved as compared to free HMFO.New knowledge for the research of MOFs immobilized enzymes and facilitates the development of immobilized biomacromolecules and synthetic biocatalysts were provided.
In recent years, cell-penetrating peptides (CPP) have provided an effective strategy for intracellular delivery of biomolecules in the biomedical field. The application of CPP in cancer treatment and disease diagnosis were focused, mainly focused on its roles and advantages in tumor targeted therapy and medical imaging diagnosis. Meanwhile, according to the characteristics of CPP in drug delivery system, the deficiency of CPP should be improved to expand the possibility of combined drug utility, which has become the research hotspot.CPP and its applications were reviewed, then describes some optimized and improved methods to expand the clinical application of CPP.
Tumor immunotherapy is aimed to inhibit the proliferation of tumor cell and kill the tumors through regulating the immunity of the body. In recent years, tumor immunotherapy has gained great progress in clinical practice, especially in the aspect of blocking the immune check point. The main methods for tumor immunotherapy are antibody therapy and adoptive cellular therapy. However, there are some shortages in the present immunotherapy, such as high side effects and high cost for treatment. Therefore, it is necessary to develop new methods that are efficient, safe and low cost. Aptamers are signal-strand DNA or RNA oligo-nucleotides obtained throughout systematic evolution of ligands by exponential enrichment (SELEX).The aptamers are similar to antibody, which can bind to their targets with high affinity and specificity. Moreover, aptamers have the advantages of low immunogenicity, penetrating tissues easily, convenient chemical synthesis and modification, and have the potential to take the similar role as the antibody for tumor immunotherapy.Presents the new applications of aptamers in cancer immunotherapy was reviewed, mainly including immune checkpoint immunotherapy, bispecific aptamer immunotherapy, aptamer-targeting siRNA immunotherapy and antibody-aptamer combination immunotherapy.
As a novel kind of nano-drug carrier, the polymersomes have the characteristics of biodegradability, stability, biocompatibility and modifiable multi-functionalization and so on. Polymersomes can be prepared by changing polymer type and the ratio of hydrophilic - hydrophobic block, which possess different morphological and membrane properties. After modifiing polymersomes, more functions can be given to realize the ability of controlling drug release and targeting drugs. The structure, composition, preparation methods and the application of drug delivery system about polymersomes have been reviewed in detail. The purpose is to know the latest research progress of polymersomes and some important problems in this field need to be solved by scientists in the future.
The functional polymer material chitosan (CS) and other composite materials have made some progress in the field of biomedicine as a scaffold material for tissue engineering. The functional groups of CS can polymerize some polymers to enhance the performance of all aspects of its composite scaffold, making it more versatile and more efficient. In nerve injury, CS scaffold material plays an important role in promoting nerve regeneration and repair,and mainly summarizes the application of CS in nerve tissue engineering.
The eukaryotic intima system consists of intercellular membranous organelles, including the formation of exosomes and autophagy, which play an important role in stress response and maintenance of cell homeostasis. Exosomes are extracellular vesicles secreted into the body by multivesicular bodies containing contents of proteins and nuclear acids, while autophagy is a process of lysosomal-dependent degradation and recycling. There is a common molecular mechanism between the formation of exosomes and autophagy, and substantial interaction between them were founded.The formation of exosomes and the process of autophagy was reviewed, including the relationship between the two and lysosomes.
Autophagy is an evolutionarily highly conserved intracellular degradation system designed to maintain cellular homeostasis in response to cellular stress. In physiological states, the level of autophagy is usually low; however, it is significantly upregulated under oxidative stress, nutritional starvation, and various pathogens. Many studies in the past have shown that autophagy plays an important role in the regulation of various histiocytes and physiological functions. Early studies have found a link between autophagy and neutrophil death, a necessary process closely related to inflammation. Autophagy plays a crucial role in neutrophil-driven inflammation and defense against pathogens in human and mouse models. Autophagy is essential for neutrophil differentiation and major functions, including degranulation, reactive oxygen species production, and release of neutrophil extracellular trapping nets. The role of autophagy in neutrophils, from neutrophils to inflammatory responses and NETosis cell death in the bone marrow were focused.
Terpenoids have considerable commercial value, but the production process is complex and the yield is low. It has become a hot spot to synthesize terpenoids from microorganism. Corynebacterium glutamicum contains a pathway to produce carotenoid, which is a natural advantage for the synthesis of terpenoids heterologously. The synthesis of terpenoids from C. glutamicum is summarized, including the terpenoids synthesis pathway in C. glutamicum, key enzymes and global regulatory mechanisms in this pathway. And the advances in this pathway in synthesis of monoterpenes, sesquiterpenes, and tetraterpenes are summarized. The problems and advice efficient synthesis of terpenoids by C. glutamicum is discussed.
In the recent years, the governments, universities, enterprises and public have paid great attention to human microbiome and health research, which aims at comprehensively and systematically analyzing the structure and function of microbiome, as well as the physiological regulation mechanisms, and providing new ideas for solving health problems. The research drives the development of probiotics and prebiotics health foods and dietary supplements, and fecal microbiota transplantation industry, and also spawns the rise of microbiome testing and health guidance, as well as microecological drug industry. This paper analyzes the status of the global human microbiome industry, and provides the main bottlenecks and future development directions of the industry, and also puts forward the specific development suggestions for the human microbiome industry of China.
