Objective: To investigate the effect of RS4651 on the EMT of hepatic cell AML12 in mice and the potential mechanism.Methods: AML12 cells were treated with RS4651 at different concentrations. Western blot and RT-PCR was used to detect the expression of E-cadherin, N-cadherin and Vimentin of RS4651 treatment groups and control group to investigate the effect of RS4651 on EMT of AML12 cells and SMAD7-Knockdown AML12 cells. RNA-sequencing identified the key node genes in the signaling pathway and the interaction network of RS4651. The proliferation and migratory effects of RS4651 treatment on AML12 cells with or without silencing by SMAD7-siRNA.Results: RS4651 could significantly upregulate the expression of E-cadherin and downregulated the expression of N-cadherin and Vimentin in a concentration-dependent manner. RNA-sequencing data showed that the target gene was SMAD7 in TGF-β1 signalling pathway. The expression of E-cadherin was relatively decreased in the SMAD7-siRNA+RS (60 μmol/L) group compared with the RS (60 μmol/L) group, while the expression of N-cadherin and Vimentin were relatively increased, and the proliferation and migration of AML12 cells were also increased.Conclusion: RS4651 can inhibit EMT, proliferation and migration of mice hepatocyte AML12 cells through SMAD7.
Objective: Ribosomal proteins (RPs) have many functions as known, including participation in regulating cell growth and responding to stress conditions. The object of this research is RpRPL22, a nodulation-related gene isolated from the legume Robinia pseudoacacia, which has a high degree of homology with the large ribosomal subunit protein RPL22 through sequence alignment. It has conducted a more in-depth exploration on how RpRPL22 regulates the infection of rhizobia and plays an important role in the process of symbiotic nodulation.Methods: Real-time fluorescence quantitative PCR technology (qRT-PCR) was used to reveal the expression level of RpRPL22 at different time after inoculation and also in different tissues. GFP reporter gene was employed to identify the subcellular localization of RpRPL22. Rapid amplification of the cDNA ends (RACE) technology was applied to get the full-length cDNA of the target gene. The RNA interference (RNAi) recombinant vector was constructed through the Gateway BP recombination technology and then transformed into Agrobacterium K599 through electrotransformation and then infect the radicle. After inoculated with rhizobia, transformed line phenotypes were observed and statistically analyzed to verify RpRPL22 function in the symbiotic nodulation process of R. pseudoacacia. To observe whether the target gene has an influence on the nodulation process from the macro level statistics, and then reveal the important function of the target gene in the symbiotic nodulation process from the molecular level.Results: Gene expression analysis showed that, with the exception of the 25th day post-inoculation (dpi), the expression level of RpRPL22 in the inoculated roots decreased compared with the non-inoculated control. In matured nodules, the RpRPL22 expression was peaked at the 25th dpi. The results of subcellular localization of both onion epidermis and hairy root showed that fluorescence signals of RpRPL22-GFP fusion were distributed in nucleus and cytoplasm under the control of 35S promoter of cauliflower mosaic virus (CaMV). The phenotypic observation results of RNAi transformed plants, such as fresh weight and effective nodulation number, were significantly lower than those of the control group. At the same time, the number of infection lines and nodule primordia was significantly lower in RNAi transformed plants than in the control. Nodule section is used to observe the microscopic and ultrastructure of nodules, which showed that the infected cells in the nitrogen-fixing zone (ZIII) in the nodules of RNAi plants were significantly reduced compared with the control group. Observation of the bacteroid morphology in a single infected cell of the root nodule by transmission electron microscope revealed that the bacteroid in RNAi nodules was severely shrunk and deformed and only a small amount of bacteroids were contained in infected cells. Besides, the fusion of multiple symbiosomes occurred and the space between the bacteroids increased. In contrast, the infected cells in the control nodules developed normally, with smooth edges and uniform cytoplasm. The above indicated that the nodule development process of RpRPL22-RNAi plants was obviously blocked.Conclusion: Ribosomal protein (RP) can participate in the regulation of the symbiotic nodulation process of leguminous plants, and the related homologous gene RpRPL22 may play an important role in initiating rhizobia infecting plants and preventing the degradation of bacteroids.
Objective: Streptomyces fradiae is the main production strain of the aminoglycoside antibiotic neomycin. Its neomycin B has strong antibacterial activity, anti-cancer and anti-HIV effects, and it is of great significance to increase the titer of neomycin B.Methods: We added different kinds and concentrations of inorganic salts to the medium to change the physical and chemical properties near the cell wall, osmotic pressure and C/N ratio of the medium under the condition of satisfying the salt ions required for the normal growth of microorganisms.Results: The degree of influence of different inorganic salts on the titer of neomycin B from high to low is (NH4)2SO4, NaCl, KCl and K2SO4 from high to low, and the titer of neomycin B reached the highest value of 15 864 U/mL when 60 mmol/L (NH4)2SO4 was added, the maximum yield of NaCl at 80 mmol/L concentration was 7 429.7 U/mL, which was 3.8 times and 0.82 times higher than that without adding inorganic salts. Then we added different concentrations of (NH4)2SO4 to the culture medium containing 80 mmol/L NaCl and sampled them regularly to detect the changes of amino nitrogen, reducing sugar, pH, TG, bacterial concentration and neomycin B titer, it was found that the consumption rate of amino nitrogen, reducing sugar and TG was increased at the concentration of 60 mmol/L (NH4)2SO4, the maximum specific growth rate of mycelium was 0.097 /h, the synthesis of neomycin B was accelerated and the yield was increased to 17 399 U/mL, at the same time, the mycelium was shortened and the sporulation was advanced.Conclusion: The salt-enhanced culture could be used as a morphological engineering method to improve the yield of neomycin B by changing the microscopic morphology of Streptomyces fradiae. It can also be used as a medium optimization strategy to increase the yield of neomycin B, which laid the foundation for further studies on increasing the yield of Streptomyces secondary metabolite.
Objective: Transposition is an effective strategy for discovering new functional genes and developing high-titer production strains. The Tn5-based transposon mutagenesis system was rationally designed and constructed, and applied to Streptomyces avermitilis to screen the engineered strains with high avermectin production.Methods: In pUCTN, two strong promoters' sequence of kasOp* and P21, commonly used in Streptomyces, were introduced into the upstream and downstream of the transposable elements, respectively, which can enhance the transcription and expression level of the genes located at the insertion position. Two transcriptional terminators T1 and T2 were also added into the upstream and downstream of the transposable elements to terminate the transcription of genes adjacent to the insertion site. The purpose of introducing promoters and terminators is to enhance the disturbance to the physiological metabolism of the mutants.Results: To improve the transposition efficiency, the ratio of donor and recipient bacteria was optimized, and 500 transposon mutants were selected and tested for avermectin production, 3 high avermectin-producing strains were screened, and the yield is significantly increased 50% than that of the parent strain.Conclusion: This Tn5 transposon mutation system established in this paper provides an effective molecular genetic tool for exploring the gene function and physiological metabolism of Streptomyces avermitilis.
Objective: The self-assembly ability of phage Qβ virus-like particles (VLPs),prepared by Escherichia coli (E. coli) prokaryotic expression system was verified. To determine their immunogenicity, New Zealand rabbits were immunized with purified Qβ VLPs, and them ability to enter mammalian cells was also detected.Methods: The pET-28-Qβ-CP plasmid was constructed, and Qβ VLPs were produced by E. coli expression system. Qβ VLPs were purified by sucrose density gradient centrifugation and gel filtration (Sephacryl S-400 column). The particle morphology of Qβ VLPs were observed by transmission electron microscopy (TEM). Antisera were obtained from the rabbits immunized with the VLPs, along with or without adjuvant, and subsequently, antibodies were purified by Protein G resin. The specificity was determined by Western blot. The indirect immunofluorescence assay (IFA) was used to detect the entry of Qβ VLPs into cells.Results: High purity Qβ VLPs were obtained. TEM results showed that substantial VLPs were observed with diameter of about 28 nm. Western blot showed that Qβ VLPs were specifically recognized by anti-rabbit polyclonal antibodies. Adjuvant had no negative effect on the production of antibody. Based on IFA, the results showed that Qβ VLPs entered various mammalian cells.Conclusion: Qβ VLPs were successfully produced in this study, and they would have great potential as carriers to facilitate the development of future vaccine based on Qβ VLPs.
Objective: The aim of this study is to develop a novel cAMP fermentation process via inhibiting phosphodiesterase activity.Methods: cAMP fermentations with aminophylline addition were conducted in a 7 L fermenter and corresponding fermentation kinetics, key enzymes activities and energy metabolism levels were analyzed. Finally, a fermentation process with aminophylline and citrate coupling added was proposed and conducted for improving cAMP content.Results: Due to 5 mg/L aminophylline added, cAMP concentration was increased by 25.9% with a decrease of 41.6% for coproduct adenosine when compared with those of a control group. There were no obvious deviation for adenylate cyclase and succinyladenylate dehydrogenase activities in the two fermentation batches whereas the activities of phosphodiesterase and 5'-nucleotide enzyme were obvious lower than those of the control group. The energy metabolism analysis results showed that intracellular ATP/AMP were lower than control with an improved AMP level, indicating that ATP synthesis had been the main limited factor for product accumulation. Using the fermentation process with aminophylline and citrate coupling addition, cAMP content achieved 4.48 g/L with 22.1% and 13.8% increasement than those of aminophylline and citrate individual added, and adenosine content was 0.98 g/L with 51.7% and 25.3% lower than individual added.Conclusion: With aminophylline added, the activities of phosphodiesterase and 5'-nucleotide enzyme were inhibited and cAMP content was also improved obviously due to decreased cAMP decomposition and adenosine synthesis. However, the ATP synthesis level had been the main limiting factor for improving cAMP production. The fermentation process with aminophylline and citrate coupling addition could decrease cAMP decomposition and improve energy metabolism simultaneously, which further promoted cAMP fermentation synthesis.
Objective: To produce benzyphetamine antigen by coupling modified benzyphetamine using the chemical method with carrier protein and its epitope was preserved.Methods: Benzafetamine artificial antigen was prepared by conjugation of carrier protein with benzafetamine hapten that was synthesized by adding active groups to a class of available chemically modified link arms by the carbodiimide method.The results were identified by UV absorption spectroscopy, SDS-PAGE and colloidal gold immunochromatography.Results: Through these tests, the Benzfetamine from this experimental scheme was successfully coupled to the carrier.This antigen had high purity,good activity and good anti-cross interference reaction,which also had high specificity with Benzfetamine antibody.Conclusion: The Benzfetamine antigen synthesized by this method can be used in immunoassay methods, and can also be used as an immunogen to prepare relevant antibodies.
Mesenchymal stem cells (MSC) play an important role in the treatment of inflammatory diseases through immune regulation and paracrine effect. The paracrine effect of MSC is mediated by secreting soble factors and releasing exosomes (Exo). Exosomes transfer DNA, proteins/peptides, mRNA, microRNA, lipids and organelles into recipient cells and display functions directly. Therefore, MSC-Exo instead of MSC provides a new strategy for the treatment of inflammatory bowel diseases. In this review, we summarize the progress of MSC-Exo derived from various tissues (bone marrow, umbilical cord and adipose) for the treatment of inflammatory bowel diseases.
MicroRNA (miRNA) is a non-coding single-stranded RNA that is widely distributed, diverse in functions, and highly conserved among species. MiRNA can regulate gene expression by completely or incompletely targeting the 3'non-coding region (3' UTR) of target mRNA, and play a regulatory role at the transcriptional level. At the same time, miRNA exists in various body fluids in a stable form, and can be used as a biomarker under different physiological or pathological conditions. Based on the early high-throughput sequencing found that miR-148 is differentially expressed in porcine colostrum and normal milk exosomes, this article intends to review the progress of miR-148 related research in terms of immunity, tumors and other biological functions, with the hope that it provides reference for the study on the biological functions of miR-148 carried by exosomes.
The nucleus is the control center of cell genetics and metabolism, regulating the cell’s responses to the outside environment, metabolism, growth and differentiation and other cellular activities. Many studies have shown that in the process of bacterial infection into host cells, some effector proteins of pathogenic bacteria enter the nucleus of the host cells, affecting gene transcription, RNA splicing, DNA repair, chromatin remodeling, etc. These nucleus-targeting pathogenic effector proteins are called nucleomodulins. This review summarized the means of these nuclear regulatory proteins entering the host nucleus, aftersecreted by pathogenic bacteria. Importantly, the functions of these nucleomodulins regulating the activities of host cells were elucidated. It provides a theoretical basis to further explore the infection and pathogenic mechanisms of intracellular bacteria on host cells.
The capsular polysaccharide (CPS) of bacteria is an important part of bacterial biofilm. It plays an essential role in the bacterial growth and division. In the meantime, this structure is related to the cell wall morphology, bacterial resistance to the external environment and immune response. In pathogenic bacteria, capsular polysaccharides act as virulence factors, so they have been studied extensively. In gram-positive bacteria, the chemical structure, biosynthesis process and functional application of capsular polysaccharides have been paid more and more attention. In this paper, the chemical structure, synthesis pathway, function and application of CPS in gram-positive bacteria were reviewed. Firstly, the bacterial distribution, chemical composition and structural specificity of the CPS from the pathogenic and non-pathogenic strains were discussed. The discussion focused on three representative Gram-positive pathogenic and non-pathogenic strains: Streptococcus pneumonia, Staphylococcus aureus, and Lactococcus lactis. Next, three main ways of the capsular polysaccharides biosynthesis in Gram-positive bacteria were reviewed: Wzx/Wzy-dependent pathway, ABC transporter pathway and synthase dependent pathway. The synthesis process and related genes of the corresponding polysaccharides were explained with examples. The physiological functions of the capsular and surface polysaccharides in gram-positive bacteria were introduced, such as barrier protection, intercellular adhesion, and participation in the immune response of host cells. In combination with the biological functions of CPS, the research progress of main applications was summarized, such as the construction of high tolerance engineering strains and the development of vaccines. Due to the differences in the characteristics of capsular polysaccharides, their chemical structure and synthesis regulation are still not clear. Finally, combined with the broad prospects in pharmaceutical research and industrial production, the prospects and suggestions are provided for the future research of bacterial capsular polysaccharides.
In recent years, due to the dual characteristics of genetic modification and traditional drugs, nucleic acid drugs have gradually attracted tremendous attention in the field of precision biomedicine and disease treatment. In order to further promote the innovation and development of nucleic acid drug industry of China, a combination of quantitative analysis and qualitative analysis have been used to analyze the situation of nucleic acid drugs of approval/authorization, market and research domestically and abroad. The results showed the global R&D status and industry development trends of four major categories of nucleic acid drugs: ASO, siRNA, RNA aptamer and mRNA. Besides, policies and measures to support the innovation and development of nucleic acid drugs in China, and the main direction of future technology and application potential have been sorted out and analyzed. Facing the urgent domestic demand for gene therapy and the severe situation in the innovation of nucleic acid drugs, countermeasures and suggestions have been put forward to promote innovation at the source, improve the mechanism for transfer and transformation of results, and create a good competitive environment.
COVID-19 that broke out at the end of 2019 swept the world. It has become a major global health and security challenge that requires the joint efforts over the world. Through hard work, China has basically controlled the domestic epidemic, and has made significant progress in related research and public health products. At the same time, China has strengthened international scientific and technological cooperation with participating countries in the “Belt and Road” Initiative.The progress of international S&T cooperation between China and related countries to combat COVID-19 in terms of basic research, research projects, etc. were summarized, and it can be seen that: China has formed a complex network of intersected and diversified nodes with the countries participating in the “Belt and Road” Initiative, and mainly with countries in Southeast Asia, Central and Eastern Europe and Western Asia; The institutes in China and countries participating in the “Belt and Road” Initiative has launched a lot of substantive research in the areas of prevention, control, epidemiology and treatment, and aid cooperation accounted for a large proportion. In the future, China should strengthen the biotechnology industry cooperation and technology transfer, give full play to the demonstration effect of the regional fulcrum countries, and build a richer, closer and more pragmatic scientific and technological cooperation relationship under the “Belt and Road” Initiative.
