Objective: To analyze the glucose metabolism pathway associated with astrocytes in Alzheimer’s disease (AD) by the bioinformatics method, and to provide a theoretical basis for revealing the glycolysis metabolism of astrocytes in the brains of AD patients. Methods: Firstly, the single-cell transcriptome data from AD patients and healthy persons were analyzed with t-distributed stochastic neighbor embedding (t-SNE); secondly, the genes differently expressed in astrocytes from AD patients and healthy persons were analyzed by Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA); lastly, the transcription cofactors related to AD were analyzed by the transcriptional regulatory network. Results: The t-SNE analysis indicated that the ratio of excitatory neurons and astrocytes was decreased in the brains of AD patients. In addition, the decreased astrocytes were characterized with RASGEF1B+SLC26A3+ or NRGN+CALM1+; GO analysis showed that differentially expressed genes were mainly related to axongenesis, neuronal migration, glial cell differentiation, zinc homeostasis, positive regulation of synaptic transmission and vascular transport. KEGG analysis showed that the differentially expressed genes were mainly related to PI3K-Akt signaling pathway, AMPK signaling pathway and calcium signaling pathway. GSEA analysis results showed that the differentially regulated genes in AD patients were enriched in glycolysis and gluconeogenesis pathway. In addition, PKM, PFKL, ACSS1 and LDHB were down-regulated in astrocytes from AD patients.Transcriptional regulatory network analysis showed that PKM,SOX2 and SOX9 were down-regulated in astrocytes from AD patients. SREBF1 and BCL6 were up-regulated in astrocytes of AD patients. Conclusion: The reduction of excitatory neurons and astrocytes in the brains of AD patients, as well as the down-regulation of the glycolysis pathway in astrocytes from the brains of AD patients, suggest that the impaired glycolysis in astrocytes could be one of the mechanisms underlying the development of AD.
Objective: To explore the functional role of XIAP-PTEN neddylation axis in colorectal cancer at cell line and clinical level. Methods: The expression level of XIAP was analyzed in tissue microarray of colorectal cancer by immunohistochemical staining, and the protein level of XIAP was detected in human colorectal cancer and adjacent normal tissues by Western blot. Co-IP was performed to analyze the interaction between endogenous PTEN and XIAP in SW480 cell line. CRISPR Cas9 technology was used to construct XIAP-knockout SW480 cell line, and the neddylation level of PTEN was analyzed in sg-XIAP cells by immunoprecipitation and Western blot. XIAP knockout or wild type SW480 cell lines were co-transfected with FLAG-Vector or FLAG-PTEN-Nedd8 plasmid, and then CCK8 and Transwell experiments were used to assay the proliferation and migration of XIAP-PTEN neddylation axis in SW480 cells, respectively. Results: The expression levels of XIAP were up-regulated in colon and rectum cancer tissues compared with adjacent normal tissues. XIAP interacted with PTEN in SW480 colon cancer cells. Deletion of XIAP inhibited PTEN neddylation in SW480 cells. The level of PTEN neddylation was elevated in colorectal cancer tissues compared with adjacent normal tissues. Deletion of XIAP inhibited the proliferation and migration of SW480 cells significantly, while PTEN-Nedd8 fusion protein rescued the phenotypes of XIAP deletion in SW480 cells. Conclusion: XIAP-PTEN neddylation axis promotes SW480 colon cancer cell proliferation and migration.
Objective: To express PSMA (prostate-specific membrane antigen) specific multivalent nanobodies in the prokaryotic system, and preliminarily evaluate their biological properties. Methods: The multivalent nanobody expression vectors were constructed by the Bglbrick method, and then transferred into E.coli Rosetta(DE3) for protein production. SDS-PAGE and Western blot assays were performed to identify the purified nanobody samples. BCA (bicinchoninic acid assay) kits were used for expression yield determination. The specific binding affinities were evaluated by immunofluorescence and flow cytometry. The binding EC50 values were detected by cell-based ELISA. The endocytosis efficiency was investigated via flow cytometry. Results: Recombinant E. coli strains for PSMA specific monovalent, bivalent, trivalent and tetravalent nanobodies production were successfully constructed, respectively. Fermentation results showed that all four kinds of nanobodies could be expressed as soluble proteins with high yields at shake flask level, among which the bivalent nanobody presented the highest expression yield [(259.14±23.56) mg/L], whereas the monovalent type lowest [(100.58±6.27) mg/L]. In the subsequent cell binding assays, results indicated that all four nanobody samples could specifically recognize and bind to PSMA-positive tumor cells. Notably, compared with monovalent nanobody, the binding affinity of bivalent, trivalent and tetravalent nanobodies were improved approximately 3.32-, 2.29- and 2.03-fold, respectively. Finally, the endocytosis experiments were conducted and the results suggested that all four kinds of nanobodies could be efficiently endocytosed by PSMA-positive tumor cells and the uptake rates in 0.5 h were all above 80%. Conclusion: PSMA specific multivalent nanobodies, especially PSMA specific bivalent nanobody, had higher expression yield and better binding affinity than monovalent nanobody. Meanwhile, they remained the same uptake level with monovalent nanobody. In this context, PSMA specific multivalent nanobodies could be a potential class of candidates for the development of PSMA-based therapies.
Populus tomentosa Carr., LM50, as an elite with characteristics of fast growth, strong stress-tolerance and no flying fluff, is usually recommended as an optimal material for genetic transformation in woody plants. The long-term asexual propagation in practice would cause the decline of excellent traits. During tissue culture, problems such as adventitious bud differentiation and rooting difficulties of explants often occur. Through anther induction approach, the negative impacts caused by the aging of the explants will be eliminated or alleviated, and P. tomentosa can be rejuvenated in a short time, providing more optimal materials for genetic transformation. Meanwhile, haploid individuals are expected to be obtained, which will be used in genomics research and ploidy breeding. Using the ‘LM50’ of the P.tomentosa gene bank in Guanxian County, Shandong Province as the test material, by comparing morphological characteristics and microspore stage, the mononuclear side-stage anthers were selected for regeneration in vitro. The effects of auxins and cytokinins in induction of callus, adventitious bud differentiation and rooting were assessed, respectively. The ploidy levels of the plants generated from anther induction were identified by the flow cytometry and chromosome counting. Furthermore, the leaves of plantlets induced by anthers as explants were used to establish a tissue culture system with high leaf differentiation rate and high rooting rate. The comparison of the microspore development period and the external morphological characteristics of the flower bud shows that most of the microspores were in the mononuclear side stage when the 1/4 inflorescence emerged from floral bud whose size was (1.98 ± 0.06) cm; the anthers in this period were selected to induce callus formation. The medium with the highest callus induction rate was H + 1.00 mg/L NAA + 1.00 mg/L BA, and the induction rate was about 28.89%. The callus was further differentiated into adventitious buds.The optimal shoot induction medium was MS + 0.05 mg/L NAA + 0.50 mg/L BA, and the induction rate was approximately 22.23%; adventitious shoots were inoculated to rooting medium. The optimal rooting medium was 1/2 MS + 0.30 mg/L IBA with 93.30% rooting rate; ploidy identification of 27 regenerated plants cultured in anthers was conducted by flow cytometry and the chromosome compression method, and the plants were all diploid. The optimal medium of in vitro regenerating for leaves and stems of plants originated from anthers was MS+TDZ 0.10mg/L + NAA 0.10 mg/L + BA 0.50 mg/L, and the regeneration rate was as high as 92.23%. The rooting medium for adventitious buds produced by leaf differentiation was the same as that for adventitious buds induced by callus, and the rooting rate was the same. The study obtained ‘LM50’ anther-induced regeneration plants, and established a leaf culture system for the anther regeneration plants, with high differentiation rate and rooting rate, which can be used for the rapid propagation of this excellent clone and genetic transformation of P.tomentosa, and laid the foundation for the molecular design and breeding of P. tomentosa.
Nicotinamide adenine dinucleotide (NAD) and its reduced form are universal redox cofactors involved in many cellular reactions. Cellular NAD level disturbance routinely leads to unexpected biological effects, and thus it is difficult to regulate a redox pathway-of-interest by manipulating NAD level. Recently, a non-natural cofactor nicotinamide cytosine dinucleotide (NCD) was devised and orthogonal redox systems based on NCD were developed, providing a new strategy for regulating cellular metabolism. To achieve more efficient regulation of lipid metabolism of the oleaginous yeast Rhodosporidium toruloides, a codon-optimized gene NCDS encoding NCD synthetase (NcdS) was integrated into the genome of R. toruloides by an Agrobacterium-mediated transformation method. Engineered strains were found with proper expression of NcdS and enzyme activity of the cell lysates reached 8.1×10-3 U/OD600 nm based on a coupled colorimetric assay. Successful biosynthesis of NCD by the cell lysates was further verified by high-performance liquid chromatography and ultra-high-resolution mass spectrometry. Intracellular NCD levels up to 41.6 μmol/L were realized upon feeding 5.0 mmol/L of nicotinamide riboside to the media used for cell culture. Results also showed that the expression of NcdS had little detrimental effect on lipid production capacity. It is expected that lipid metabolism may be reconstructed and regulated by NCD upon further expression of other NCD-preferred enzymes in R. toruloides.
Antibody drug conjugates (ADCs) are a type of novel anti-tumor drugs, which are composed of three components: antibody, cytotoxic drugs and linker. Compared with traditional cytotoxic drugs, ADC has the ability to specifically target tumor cell and release small molecular drugs to achieve the effect of tumor-specific killing, showing good therapeutic potential in clinical practice. Particularly, the antibody of ADC can accurately deliver small molecule cytotoxin to the tumor by combining with targeted antigens on the surface of tumor cell, which is one of the core elements affecting the efficacy of ADCs. The review outlines advances in the study of antibody composition and targets of ADC drugs.
Diabetes is the third most harmful disease to human health after cancer and cardiovascular disease. Treatment for type 1 or type 2 diabetes requires daily injections or continuous infusion of exogenous insulin to regulate blood sugar in the body to normal levels. However, current insulin treatments are limited by the risk of hypoglycemia. Through the application of a delivery system based on biomaterial carriers, the bioavailability of insulin can be improved while the occurrence of adverse reactions can be reduced.Therefore, the research and development based on an intelligent insulin delivery system is necessary to improve the controllability of insulin administration. This paper reviews different methods of insulin delivery in recent years. The mechanism of an intelligent insulin delivery system was described, and the research status and existing problems of intelligent insulin delivery systems under different drug delivery methods were discussed.
Cellulose is a low-cost and renewable resource with abundant reserves, but it is difficult to be used because of its compact structure. Currently, the degradation of cellulose requires the cooperation of a variety of cellulases, but the high cost and the difficulty of re-use of free cellulase have limited its wide application. Yeast surface display technology can display multiple cellulase enzymes on the cell surface after being fused with the anchoring protein, so that a yeast surface display cellulase system could be constructed. This system can degrade cellulose efficiently. On the one hand, it has the advantages of surface display, such as easy recycling, good stability, simple operation, and low cost; on the other hand, it can also degrade cellulose into glucose effectively, and has the potential to produce bioethanol by metabolism. The paper summarizes the construction principles of a yeast surface display system, and divides yeast surface display systems into direct display systems and indirect display systems according to the different anchoring methods of exogenous proteins. The direct display system is that the exogenous protein is directly connected to the cell through the anchor protein. Indirect display systems are linked to cells through exogenous proteins by means of scaffolds. Factors affecting the efficiency of the display system mainly include cell type, anchor protein, scaffold, environmental factors and promoter type. The surface display system consists of GPI system, Pir system and FlO1 system according to different anchor proteins. The yeast surface display cellulase system can ferment cellulose to produce ethanol, and a lot of progress has been made so far, which provides a reference for the construction of an efficient yeast surface display cellulase system and other multi-enzyme systems. The review describes the construction principles of a yeast surface display system, summarizes the influence of major factors on the display system, and introduces the application of this technology in the degradation of cellulose. It provides guidance for the construction of high-efficiency yeast surface display of cellulase and other multi-enzyme systems.
Outer membrane vesicles (OMVs) are membrane vesicles with a diameter of 20-300 nm secreted during bacterial growth. They are composed of phospholipids, lipopolysaccharides, proteins, RNA or DNA and so on. OMVs contain a large number of bacterial antigens, which enhance the expression of cytokines and costimulatory molecules by initiating signal transduction pathways, promote antigen presentation and effectively activate the immune system. The virulence factors encapsulated in OMVs can be transmitted to host cells, stimulate the interaction between bacteria and host cells, and have inherent anti-tumor activity. OMVs are conducive to engineering design, and also can be used as an efficient drug delivery carrier to achieve the combination of immunotherapy and chemotherapy-phototherapy, so as to improve the anticancer ability of drugs. They have a good prospect in tumor immunity, tumor engineering vaccine and drug loading, and are considered to be a new means of anti-tumor therapy. This paper summarizes the research progress of bacterial outer membrane vesicles in tumor therapy from the aspects of structure and components, formation mechanism and anti-tumor mechanism, so as to provide reference for the further study and clinical application of bacterial outer membrane vesicles in the future.
To evaluate the immune protection of recombinant SARS-CoV-2 S1 and S protein vaccine. Methods: Recombinant SARS-CoV-2 S1 or S protein combined with aluminum hydroxide adjuvant was inoculated at different doses of 0.1 μg, 1 μg, 5 μg and 10 μg per mouse for 6-8 weeks. Serum IgG antibody titers were detected by enzyme linked immunosorbent assay (ELISA) after second immunization. The serum neutralizing antibody titers of the immunized mice against pseudotype SARS-CoV-2-Fluc WT, B.1.1.7, P.1, B.1.617.2, B.1.621, 501Y.V2-1 strains were compared by pseudovirus neutralization test. The cellular immune levels of sera were detected by enzyme-linked immunospot assay (ELISpot).Results: Both SARS-CoV-2 S and S1 proteins could induce strong IgG antibody levels in mouse model. The sera of mice immunized with S1 protein showed obvious neutralization activity against SARS-CoV-2-Fluc WT, B.1.1.7 and P.1. The sera of mice immunized with the recombinant S protein also showed obvious neutralization activity against SARS-CoV-2-Fluc B.1.617.2 in addition to SARS-CoV-2-Fluc WT, B.1.1.7 and P.1. The serum of mice immunized with two kinds of proteins had the strongest neutralizing effect on SARS-CoV-2-Fluc WT. Mouse spleen cells immunized with S protein could significantly induce the production of interferon-γ (IFN-γ) and interleukin-4 (IL-4). The levels of IgG antibody, neutralizing antibody and cellular immunity induced by S protein were higher than those of S1.Conclusion: Recombinant SARS-CoV-2 S protein vaccine can induce protective immune responses.
Objective: Hepatitis B virus core protein HBc was used as vector to construct virus-like particles expressing novel coronavirus spike protein receptor binding domain RBD, and their immunogenicity was identified, which provides a new idea for the development of COVID-19 vaccines. Methods: The amino acid coding sequence 78 and 81 of hepatitis B virus core protein HBc (1-183 aa) were inserted into novel coronavirus spike protein receptor binding domain RBD and ligated by flexible linker (G4S) 3. After sequence optimization, the fusion gene was cloned into prokaryotic expression vector pET-28a (+) and transformed into expression strain Rosetta. After induced expression in self-inducing medium, the virus-like particles (VLPs) were purified by sucrose density gradient centrifugation. VLPs were detected and identified by SDS-PAGE, Western blot and transmission electron microscope. BALB/c mice were immunized subcutaneously with the prepared VLPs in equal proportion with adjuvant. The specific antibodies in the serum of the mice were analyzed by ELISA to verify the immune effect of HBc-RBD VLPs. Results: Escherichia coli can express partially soluble VLPs in self-inducing medium. VLPs could be observed by transmission electron microscope after purification by sucrose density gradient centrifugation. Mice immunized with HBc-RBD VLPs produced specific antibodies against RBD antigen. Conclusion: VLPs displaying RBD antigen were successfully expressed in prokaryotic expression systems, and their immunogenicity was preliminarily verified by mouse experiment, which provides a new direction for the research and development of novel coronavirus vaccines.
Since the outbreak of novel coronavirus disease in late 2019, it has been a global public safety emergency to efficiently prevent and control the epidemic. Vaccine is one of the means to effectively prevent the virus from infecting humans, protect high-risk groups from rapid disease progression and minimize further spread of the virus-caused epidemic. Subunit vaccine is a safe and effective strategy that contains recombinant protein antigens of specific viral components and vaccine adjuvant that helps increasing the immunogenicity of the antigen. Since the specific immunogenic viral antigen can activate the immune system, which thus produces antibodies against immunodominant epitopes on the surface of the protein antigen, it offers subunit vaccine a high degree of protection and safety. The major severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) subunit vaccines that have been marketed and are currently in the clinical stage are reviewed. The design concepts of various antigens and types of vaccine adjuvants, the protective capacity, and the research progress of subunit vaccine candidates are introduced. The applications and technical advantages of subunit vaccine are analyzed. This review is expected to provide suggestions for subunit vaccine development and global epidemic prevention and control.
Human genetic resources refer to materials such as organs, tissues, cells and other materials that contain human genome, genes and other genetic materials, and the data they generate. Human genetic resources in China are extremely rich. It is significant for promoting life science, biomedicine and clinical research to utilize human genetic resources reasonably in China. In order to effectively protect, manage and utilize human genetic resources in China, the management department strictly follows the laws and regulations to execute administrative approval of human genetic resources. In this paper, the status of administrative license for collecting human genetic resources in 2021 in China is summarized. Then, the practical work is combined to analyze the existing problems. Finally, some recommendations are made for the management of human genetic resources in China.
With the increasing exhaustion of global resources, various countries have explored bioeconomy as an economic model that can cope with environmental, climate, resource problems and food security crisis. China recently released the “14th Five-Year Plan for Bioeconomy Development”, raising the bioeconomy to the level of national strategic development for the first time. Based on the innovation of life science and biotechnology, emerging industries have come into being in bioeconomy including biomedicine, bioagriculture, biomanufacturing and bioenergy. Bioeconomy is an economic development model with great potential for sustainable development in the future. This paper summarizes the evolution law of the global bioeconomy, the development of the bioeconomy worldwide and the industrial development of Chinese bioeconomy. Moreover, under the complex situation in a time of unprecedented global changes in a century and the COVID-19 epidemic, the relevant countermeasures to cope with challenges and suggestions on Chinese future bioeconomy development are put forward.
Since the concept of bioeconomy was proposed, the United States, the European Union, the United Kingdom and Japan have been actively involved in roadmap planning and related project deployment in order to cultivate new economic growth areas and have the initiative in future international competition. With the rapid development of innovation and industry in the biotechnology sector in recent years, bioeconomy is expected to play a more prominent role and contribute to the national economy, leading the way to the new round of economic transformation. In order to better understand the current situation and scale of the development of the global bioeconomy, this paper selects several representative countries/regions such as the US, the EU and Japan to conduct research on the methods of bioeconomy measuring. On the basis of the definition of bioeconomy, a framework for measuring the bioeconomy in China was proposed and preliminary results were obtained to provide scientific data support for China’s bioeconomy development decisions. Finally, the paper forecasts the future scale of China’s bioeconomy, and also discusses improvements to the measuring methodology.
The year 2019 marked South Africa’s 22 years of commercial cultivation of biotech crops, planting a total of 2.68 million hectares on the three principal genetically modified crops: cotton, maize, and soybeans. South Africa is ranked 8th among all the countries that plant genetically modified crops in the world, and is the biotechnology leader on the African continent. For such a country with a super-high application rate of genetically modified crops, there is a lack of a systematic introduction to the process and current situation of its genetically modified crops commercialization now. Using the reports and other data from the International Service for the Acquisition of Agri-biotech Applications (ISAAA) from 1996 to 2019, this paper introduces the approval and cultivation of genetically modified crops in South Africa, independent research and development, management system, import and export situation and public acceptance. Based on this, some suggestions for our regulation on genetically modified crops in China are proposed.
