Objective: Endothelial cells (ECs) together with pericytes, microglia, astrocytes and neurons, form a neurovascular unit (NVU) that constitutes the blood-brain barrier (BBB). However, the role of microglia in the maintenance of the BBB remains under debate. Protein arginine methyltransferase 5 (PRMT5) is the main catalyzing arginine symmetric dimethylation methyltransferase in vivo. Our previous work found that cerebral vascular EC-specific Prmt5 gene knockout resulted in severe cerebrovascular lesions and impaired BBB. Whether microglia are activated in cerebral vascular lesions caused by Prmt5 gene knockout in cerebral vascular ECs is investigated, and microglia’s effects on the BBB permeability are explored. Methods: SP-A-Cre transgenic mice were bred with Prmt5fl/fl conditional gene targeting mice, the knock-in mice with Rosa26tdTomato (RFP) reporter lines, to generate cerebral vascular ECs Prmt5 gene knockout (Prmt5fl/fl) mice. To examine whether microglia are activated in the cerebral vascular ECs Prmt5fl/fl mice, Ionized Calcium Binding Adaptor Molecule 1 (IBA1) expression levels in the cortex, thalamus and cerebellum of the mice in the control group and Prmt5fl/fl mice were detected by immunofluorescence and Western blot. The M1 microglial markers of the cluster of differentiation 86 (CD86), the cluster of differentiation 16 (CD16), tumor necrosis factor alpha (TNF-α) and M2 microglial markers of chitinase-like protein 3 (Chil3/Ym1), arginase 1 (Arg1), and Interleukin-10 (IL-10) were detected by real-time quantitative PCR and Western blot to evaluate the microglial polarization in different brain regions. CSF1R inhibitor PLX5622 was intraperitoneally injected into the mice in the control group and Prmt5fl/fl mice to deplete microglia. IBA1 expression level was detected by immunofluorescence and real-time quantitative PCR to evaluate the efficiency of microglial depletion. Sulfo-NHS-Biotin was intraperitoneally injected into the mice in the control group and Prmt5fl/fl mice to examine the BBB integrity. Results: The expression level of IBA1 increased significantly in the cortex, thalamus and cerebellum. The IBA1 and CD68 double positive cells accumulated in the thalamic lesion area of Prmt5fl/fl mice showed that knockout of Prmt5 gene in cerebral vascular ECs led to activation of microglia. The expression of M1 markers (CD16, CD86 and TNF-α) and M2 markers (Ym1, Arg1 and IL-10) were up-regulated in the cortex, thalamus and cerebellum in cerebral vascular ECs of Prmt5fl/fl mice. PLX5622 treatment resulted in microglial depletion with a depletion efficiency of more than 70%. Depletion of microglia leads to increased BBB permeability of Prmt5fl/fl mice. Conclusion: Deletion of Prmt5 in cerebrovascular ECs leads to activation of microglia, which may be involved in the maintenance of the BBB integrity.
Objective: To develop a bispecific antibody drug conjugate (BsADC) targeting TPBG×EGFR and to investigate its anti-tumor activity in vitro and in vivo. Methods: Fully human antibodies targeting the oncofetal trophoblast glycoprotein (TPBG, also known as 5T4) and the epidermal growth factor receptor (EGFR) were acquired from the RenLite platform, which is a novel mouse model expressing the entire human antibody variable region of the heavy chain and a specific common light chain. Anti-TPBG and EGFR antibodies were assembled into anti-TPBG× EGFR bispecific antibody (BsAb) by the knobs-into-holes (KIH) technique. Structural and functional characterization of anti-TPBG×EGFR BsAb was screened by Flow Cytometry (FCM), Surface Plasmon Resonance (SPR) and High Performance Liquid Chromatography (HPLC). Anti-TPBG×EGFR BsAb and the microtubule protein inhibitor MonoMethyl auristatin E (MMAE) were assembled using the cysteine coupling strategy to generate anti-TPBG×EGFR BsADC with a drug to antibody ration (DAR) of 4. Furthermore, the in vitro cell killing and in vivo anti-tumor activity of TPBG×EGFR BsADC were explored in human-derived tumor cell lines and cell derived xenograft (CDX) models. Results: According to the GEPIA2 database, TPBG is co-expressed with EGFR in a variety of tumours. Immunofluorescence analysis of five Patient-Derived Xenografts (PDX) showed that TPBG and EGFR are co-expressed in tumour cells in varying proportions. The bispecific antibodies targeting TPBG and EGFR with high-purity were successfully developed by the RenLite co-light chain fully human antibody discovery platform. In vitro results showed that by simultaneously targeting both TPBG and EGFR on the surface of tumor cells, the bispecific antibody significantly enhanced the binding affinity (avidity), endocytosis and killing of tumor cells compared to the TPBG parental monoclonal antibody. Moreover, in the A431 (EGFRhigh/TPBGlow) model, anti-TPBG×EGFR BsADC showed stronger anti-tumor activity than the parental ADCs of TPBG and EGFR in vivo, demonstrating synergistic effects. In addition, in the NCI-H292 (EGFRmoderate/TPBGlow) and DU145 (EGFRlow/TPBGlow) models, anti-TPBG×EGFR BsADC also showed strong anti-tumor activity. Conclusion: These results suggest that combining TPBG with EGFR, a rapid internalizing ADC target, is a powerful strategy for enhancing anti-tumor activity of ADCs targeting TPBG.
By combining and screening several mutations of glutamate decarboxylase (GadB) derived from E. coli, a combined mutant M2 with a wider pH range, higher catalytic activity and higher pH and thermal stabilities was obtained. Compared with the wild-type GadB-WT, the pH range of the combined mutant M2 was effectively broadened, and it produced a higher catalytic activity of 113.43% over the wild-type GadB-WT under pH6.0. After the optimization for fermentation medium and induction conditions of the recombinant bacteria, an overall 104.13% increase of enzyme activity was gained over the unoptimized medium. On this basis, the enzymatic properties of M2 were determined. The optimum pH was 5.0 and the optimum temperature was 37℃. Compared with wild-type Gad-WT, the pH stability and thermal stability of M2 were enhanced to some extent. The kinetic parameters of M2 were determined as follows:Km was 7.316 μmol/L, kcat was 13.387 s-1, and kcat/Km was 1.830 L/(s·μmol). The combined mutant M2 obtained in this study further enriches the GAD mutant enzyme library for the synthesis of γ-aminobutyric acid and has a promising application prospect.
To express levansucrase in Bacillus amyloliquefaciens 018 (G3) efficiently. Four levansucrase genes lsLich, lsAmy, lsSub and lsMega from different Bacillus species were heterologously expressed, and five signal peptides with relatively high levels of alkaline protease identified by the research group were screened and combined. lsLich derived from Bacillus licheniformis RN-01 had the highest enzyme activity in the recombinant strain G3/pLY-2-lsLich, with an enzyme activity of 62.73 U/mL. LS-Lich was used as the target protein to screen single signal peptide and double signal peptide. The recombinant strain G3/pLY-2-SDA-ls combined with SPDacB and SPAmyE had the highest enzyme activity, and the extracellular enzyme activity reached 125.76 U/mL. Compared with the recombinant strain G3/pLY-2-SD-ls and G3/pLY-2-SA-ls, they increased by 31.3% and 39.2%, respectively, and increased by 100.49% compared with the original strain. The results indicated that the double signal peptide was helpful in increasing the secretion of LS-Lich compared with the single signal peptide, and the combination order of signal peptides also produced some differences.
Objective: Ectoine, a compatible solute, is widely distributed in halophilic bacteria to resist extreme environmental conditions. The aim of this study is to increase the accumulation of ectoine in wild-type Halomonas campaniensis sp. XH26. Methods: Magnetic nanoparticles (Fe3O4 NPs) were used, and single factor analysis, Plackett-Burman design and the response surface method were performed to evaluate the intracellular accumulation of ectoine and bacterial growth. The feasibility of nano-metal application in Halomonas fermentation was further discussed. Results: According to single factor analysis, Fe3O4 NPs can promote the strain growth and accumulation of ectoine. The optimal period for adding Fe3O4 NPs was the logarithmic growth stage of the strain. Plackett-burman and the response surface results revealed that ectoine accumulation of the strain reached 640.28 mg/L in shaker fermentation, under the optimized conditions(Fe3O4 NPs: 0.05 g/L; NaCl: 1.53 mol/L; MSG: 0.03 mol/L), which was 63.61% higher than that of the wild strain (391.35 mg/L). Transmission electron microscopy indicated that Fe3O4 NPs accumulated on the surface of bacterial cell membrane, which may play a catalytic role through surface adsorption. Conclusions: In summary, Fe3O4 NPs can effectively promote the accumulation of ectoine, and the combination of Plackett-Burman and the response surface method can better optimize the fermentation conditions of the strain. This study provides a new technical idea and reference for the subsequent application of magnetic nano-metal particles in the industrial production of ectoine by fermentation.
There was a global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. The pandemic of corona virus disease 2019 (COVID-19) caused by SARS-CoV-2 shows high infectivity and fatality rate, which has caused a great burden on human health and economic development. Vaccination is an important way to prevent and control the prevalence and spread of SARS-CoV-2. A lot of vaccines are developed and applied to prevent and control it, such as inactivated viruses vaccines, recombinant subunit protein vaccines, adenovirus vector vaccines and messenger RNA (mRNA) vaccines. The mRNA is a new drug model, which can use the body’s own translation system to express proteins with different functions. Therefore, it can be used in the treatment of many diseases, which is also considered to be a substitute for DNA and recombinant protein mediated therapy. With mRNA synthesis, purification and modification in vitro, scientists find that the mRNA is easy to be degraded due to its instability, which leads to lower transfection efficiency. Therefore, it is necessary to fabricate and develop a suitable delivery system for improving its stability and translation efficiency. The successful delivery system makes mRNA drugs attract more and more attention in cancer treatment, infectious disease prevention, protein replacement therapy and gene editing. Until now, many delivery carriers have been designed and evaluated including dendrimers, liposome, nano-emulsions and polymer nanoparticles. In addition, mRNA vaccines show such excellent characteristics as simple preparation, short development and production cycle and little cytotoxicity. Most importantly, mRNA vaccines are easy to scale up. All these advantages result in mRNA vaccines’ suitablity to deal with infection outbreaks. Here we will review the mRNA vaccine, the mechanism of action, the delivery vector and the administration ways of the mRNA vaccine, in order to provide reference for the mRNA vaccine research and development.
In recent years, due to the ravages of the COVID-19, the development of single-injection vaccine has attracted much attention. Traditional vaccine preparations need to be inoculated repeatedly for a period of time to produce enough neutralizing antibodies. In order to reduce the number of vaccination shots and improve people’s vaccination compliance, polymer materials have gradually entered people’s field of vision. Among them, Poly lactic-co-glycolic acid (PLGA) is one of the most studied and widely used polymer materials. As a synthetic polymer material, PLGA is easy to prepare and relatively low in price, and it has favorable sustained release characteristics and has good biosafety and histocompatibility. It has been approved by the U.S. Food and Drug Administration (FDA) as a drug delivery system, but it is in the ascendant in vaccine research and development. Based on the current research progress, this article summarizes the basic information of PLGA adjuvants and the related factors affecting their sustained release effect and immunomodulatory effects, so as to provide some ideas for subsequent vaccine preparation and research.
Extracellular vesicles (EVs) are lipid vesicles naturally secreted by cells. They play an important role in communication of information in physiological and pathological processes. Mesenchymal stem cells are pluripotent stromal stem cells from a wide range of sources. The potential of mesenchymal stem cell regeneration and its ability of immunomodulation have shown great promise in the repair in and treatment of lung diseases. Mesenchymal stromal cells-EVs (MSCs-EVs) have the same functional characteristics as MSCs, and many active factors carried by MSCs-EVs have shown good therapeutic effects in lung tissue, lung microenvironment and lung diseases. In this paper, the biological characteristics of MSCs and MSCs-EVs were summarized, and the mechanism and clinical application of MSCs-EVs in pulmonary diseases were discussed.
The human intestinal microbiome plays an important role in maintaining human health and the occurrence and development of diseases. The world is faced with some serious infectious diseases such as the novel coronavirus epidemic, and foodborne diseases, pathogenic drug-resistant “superbugs” and other problems are becoming more serious, threatening human health. Therefore, it is of great practical significance to use patent big data to carry out the analysis of the research and development (R&D) situation of the global intestinal microecology industry based on the patent map, make a preliminary judgment on the development direction of the intestinal microecology industry, and clarify the R&D hotspots. Based on the patent map, this paper focuses on analyzing the patent application trend, disclosure of applicants’ nations, inventor, technical composition, R&D hotspots, core patent technology path evolution trend and competitive influence of key institutions from 2011 to 2021 and from the dimensions of patent management map and patent technology map. The study found that the number of patent applications in the global intestinal microecology industry showed a gradual and rapid growth trend, reaching the second peak in 2020, and will continue to increase rapidly in the future. China is the country with the highest proportion of patent applications, acceptance and disclosure, and the top 10 first inventors (designers) in terms of application volume are mainly from China. The field of bacteroides|bacteria is a global R&D hotspot, the R&D hotspots of various countries have their own merits, and the overall complementary advantages have been formed.
Objective: To provide countermeasures and suggestions for the high-quality development of the biomedicine industry in Chongqing. Methods: Through literature, policy, news, and other network reports, the development trend and general characteristics of the biomedicine industry were summarized, and the general situation, achievements, advantages, deficiencies, and weaknesses of the development of the biomedicine industry in Chongqing were analyzed. This paper provides specific countermeasures and suggestions for such problems as talent shortage, the undustry lagging behind in transformation of innovative achievements, weak industrial competitiveness, the untapped advantage of traditional Chinese medicine resources, and a lack of leading enterprises. Results: The construction of the biomedicine industrial parks in Chongqing is focused on cultivating and supporting leading enterprises, building a multi-level investment support system, building industry-university-research community and improving community atmosphere, and strengthening multi-chain integration and development of industrial chain, capital chain, innovation chain, and talent chain.
In May 2022, China issued the 14th Five-Year Plan for the Development of Bio-economy, aiming to achieve 22 trillion yuan of total bio-economy output by 2025. The development track of China’s bio-economy has shown comparative advantages of Yangtze River Delta region, Pearl River Delta region and Bohai Bay Rim region, and Sichuan and Chongqing areas have a strong momentum of catching up. Tianjin is located in the economic center of Bohai Bay Rim region and it is an important hub connecting the three Northern regions and Northeast Asia. With unique geographical advantages and profound industrial foundation, Tianjin continues to rank among the top 50 cities in terms of scientific research and metropolitan areas in the world. The development situation of biotechnology and related industries in Tianjin was deeply analyzed based on innovative resources, distribution of biotechnology industry and policy environment in Tianjin. Tianjin is rich in innovation resources of biotechnology and related industries, which is competitive on a national scale. In particular, it has strong innovation strength on research and development. It has initially formed a ‘1+5’ regional agglomeration pattern, and the overall industrial development is on the rise. At the same time, problems were analyzed, such as fierce competition among local governments, inactive capital investment and financing, and insufficient effectiveness of local technology transfer. Countermeasures and suggestions were put forward from the aspects of accelerating the cultivation of high growth track, actively exploring the integrated development mode of science and technology and capital, and further strengthening the in-depth cooperation between industries, universities and research institutes.
The Commission on Science and Technology for Sustainable Development in the South (COMSATS) is an important intergovernmental organization among developing countries with 27 member states. China is one of the founding members. This article investigates the policy environment, resource endowments, research institutions in the area of industrial biotechnology of the member states, the research and development (R&D) progress and application status in various areas. Based on the results, we propose suggestions to deepen the cooperation between China and other COMSATS member countries for mutual benefit.
