Objective: Bladder cancer cell lines and organoid models with FGFR3 mutations were constructed at different sites first, and then their sensitivity to different tyrosine kinase inhibitors targeting FGFR3 gene mutations was determined. Methods: Cell line models with wild type FGFR3, point mutant FGFR3 (S249C, R248C and Y373C) and FGFR3-TACC3 fusion were established. Eight tyrosine kinase inhibitors were then used to test for differences in drug sensitivity in different stable cells. The phosphorylation level of FGFR3 downstream pathway protein was detected by Western blot analysis to analyze the mechanism of the difference in tyrosine kinase inhibitor (TKI) sensitivity in different stable cells. Organoid models were constructed using bladder cancer patient tissues, and the above experiment was repeated. The sensitivity of different mutant bladder cancer organoids to different TKIs was determined at the organoid level. Results: FGFR3 point mutation R248C and FGFR3-TACC3 fusion increased by 5-10 fold compared with wild type FGFR3 to half of tyrosine kinase inhibitors at the cell line level (P<0.05). Organoids derived from tumor tissue of bladder cancer patients were successfully constructed. This confirmed that the organoid has the organizational and genetic characteristics of the tumor in situ. The FGFR3 point mutations S249C and Y373C did not show significant differences in sensitivity, which can be 1-5 fold different from the wild type. The FGFR3-TACC3 fusion and the FGFR3 point mutation Y373C can significantly alter the sensitivity of cells to drugs at the organoid level. Conclusions: This study successfully constructed cell lines and organoids with different FGFR3 mutations, further demonstrating that different FGFR3 mutations have different sensitivities to tyrosine kinase inhibitors, and that organoids with the FGFR3 point mutation R248C have high sensitivity to all TKI drugs.
Objective: Interleukin-1 receptor accessory protein (IL-1R3) is a potential new target for inflammation regulation. The aim of this study is to obtain anti-IL-1R3 antibodies with cross-binding activity to human and mouse IL-1R3 and to lay the foundation for the new drug development and pharmacodynamic mechanism research of a novel inflammation intervention strategy. Methods: Based on the sequence and structure alignment of human and mouse IL-1R3, a phage-displayed human single-chain (scFv) antibody library was challenged by alternately coating immune-tubes with human or mouse IL-1R3. The variable region genes of the resulting antibodies were cloned into eukaryotic expression vectors to prepare antibodies. The binding activities of the candidate antibodies were determined by ELISA and SPR and their functional activities were evaluated by cell assay. A strategy of re-pairing the light and heavy chains of these antibodies was then followed to obtain new antibodies with improved properties. Results: Five antibodies with cross-binding activity to human and mouse IL-1R3 were identified, four of which showed comparable affinity to both human and mouse IL-1R3, approximately 10-7 mol/L. Two candidate antibodies with favorable solubility effectively blocked IL-1R3-mediated activities in the ex vivo functional assay. A new antibody with re-paired light and heavy chains showed improved binding activity and apparently increased solubility. Conclusions: By introducing antibodies AET1907 and 4H6L with comparable human-mouse IL-1R3 cross-binding activity, this study has laid the groundwork for further investigation.
Sugarcane ratooning ability is directly related to its production cost and cultivation efficiency. Sugarcane ratooning productivity is influenced by variety, environment and cultivation measures, but variety is the most critical factor. There are few reports explaining the differences in sugarcane ratooning ability at the molecular biological level both domestically and internationally. In this paper, transcriptome sequencing was used to analyze the differences in gene expression between sugarcane GR2 and ROC22 of different root years. The results showed that 100 558 transcripts and 25 582 unigenes were obtained by transcriptome sequencing. By functional annotation of unigenes, including comparison with NR, Swiss-Prot, KEGG, COG, KOG, GO and Pfam databases, a total of 53 790 unigene annotation results were obtained. The GO functional annotation was divided into three categories and 51 subcategories. A total of 1 029 differential genes were annotated in six-year-old sugarcane, and 3 391 differential genes were annotated in three-year-old sugarcane. There are eight major KEGG metabolic pathways, which are mainly involved in plant hormone signal transduction, starch and sucrose metabolism, glycine, serine and threonine metabolism, phenylpropanoid biosynthesis, homologous recombination, DNA replication, mismatch repair and plant pathogen interaction. Six abscisic acid-related differentially identified genes are: abscisic acid-insensitive 5-like protein 2 (ABI2), putative bZIP transcription factor superfamily protein, protein abscisic acid-insensitive 5 (ABI5), aba responsive element-binding factor 1 (partial ABF1), G-box binding factor 4 (GBFs), and aba responsive element-binding factor 1 (ABF). The above differential genes will serve as reference genes for our later gene expression and function analysis, and further analyze the molecular mechanism affecting sugarcane ratooning ability by combining proteomics.
In order to investigate the influence of the Sec secretion pathway on alkaline protease production in Bacillus licheniformis, the molecular chaperone-blocking protein gene hrcA and the three extracellular protease genes epr, bpr, and vpr in the genome of B. licheniformis TCCC11470 (BLΔuppΔepsΔpgs) were sequentially deleted. By comparing the alkaline protease activities before and after gene deletion, it was found that the knockout strains TCCC11470ΔhrcA and TCCC11470ΔhrcAΔeprΔbprΔvpr reached alkaline protease activities of 18 521.2 U/mL and 20 048.5 U/mL after 42 hours, respectively, which were 27.9% and 38.5% higher than the control strain BLΔuppΔepsΔpgs (14 478.6 U/mL), respectively. These results indicate that optimizing the Sec secretion pathway can effectively enhance the enzymatic activity of alkaline protease, providing new insights and research strategies for the construction of industrial enzyme production hosts.
Objective: To screen nanobodies that bind to carcino-embryonic antigen (CEA) with high affinity for the development of rapid detection methods for CEA in the future. Methods: Using human CEA protein to immunize Bactrian camels, lymphocyte cDNA was used as a template, and nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody (VHH) gene, ligated to pMECS vector, and electroporated into TG1 E. coli to construct a VHH cDNA library. After infection with helper phage M13K07, a phage display library was generated, and VHH clones bound to CEA were enriched by biopanning in ELISA, nanobodies with high binding to CEA were screened by periplasmic soluble ELISA (PE-ELISA), and IPTG-induced expression and affinity purification were performed in WK6. The binding specificity of the nanobodies to CEA was by measured by ELISA,and the epitope overlap was detected by competitive ELISA to screen paired nanobodies for the construction of sandwich ELISAs. Results: A VHH library with a size of 2.8×108 cfu was constructed. Colony PCR showed that VHH insertion was about 100%. A total of 90 clones enriched in the 2nd-3rd round were randomly selected for PE-ELISA detection, and 45 clones were positive. Sequence analysis showed that they encoded 7 nanobody sequences. In WK6, these nanobodies are expressed in a soluble form, and recombinant nanobodies with a purity close to 90% are obtained by Ni affinity chromatography. Indirect ELISA showed that all 7 Nbs specifically bound CEA, and 2 nanobodies 3G2 and 3F4 had high affinity and concentration-dependent binding to CEA, and did not bind to the unrelated protein lysozyme even at high concentrations (5 μg/mL), showing high specificity. Competitive ELISA results showed that 3G2 and 3F4 did not compete in binding to CEA, indicating that 3G2 and 3F4 bind to different epitopes of CEA antigens. Using 3F4 as the capture antibody and 3G2 as the detection antibody, the double nanobody sandwich ELISA method (referred to as 2Nbs-ELISA) was established, and its limit of detection (LOD) for CEA in solution was 1.8 ng/mL. Conclusion: The two CEA-specific Nbs obtained in this study were used to establish a sandwich ELISA method with higher sensitivity and specificity for the detection of CEA.
Interleukin-5 (IL-5), a homodimeric cytokine, is an important regulator of eosinophil (EOS) proliferation, activation and maturation. Anti-IL-5 monoclonal antibodies block the binding of IL-5 to the IL-5 receptor subunit alpha (IL-5Rα) and have been used successfully in the treatment of eosinophilic asthma. Currently available monoclonal antibody drugs require repeated administration by injection, which has a significant impact on patient compliance, and the systemic exposure rate of injection is high. To obtain nanobodies suitable for inhalation administration, monoclonal clones were selected through three rounds of panning in the natural alpaca library by phage ELISA screening. A total of 461 positive clones were obtained, of which 50 clone sequences were unique, and 30 molecules were selected for recombinant expression and purification of nanobodies. The in vitro activity of the candidate antibodies was tested by ELISA binding, ELISA blocking, FACS blocking and TF-1 proliferation inhibition assays, and a nanobody AIL-A96-Fc with the ability to block the binding of IL-5 and IL-5Rα was successfully obtained. ELISA binding assays with human and cynomolgus IL-5 showed that the molecule has good human-cynomolgus cross-species activity, and AIL-A96-Fc showed good blocking effects in FACS and ELISA blocking assays. This study not only provides a candidate nanobody (AIL-A96-Fc), but the development methodology also provides guidance for the subsequent development of additional candidate nanobodies targeting IL-5.
Objective: Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, plays a major role in the pathogenesis of Parkinson disease. Exosomes, vesicles with a diameter ranging from 30 to 200 nm secreted by cells, are considered potential carriers for drug delivery across the blood-brain barrier. To achieve efficient encapsulation of tyrosine hydroxylase mRNA in exosomes (TH-Kt-Exo), the binding properties of the archaeal ribosomal protein L7Ae and the Kt loop are exploited to enable delivery of mRNA-based therapeutics across the blood-brain barrier. Methods: The method involved constructing a recombinant plasmid containing TH mRNA with the Kt loop and a recombinant plasmid, pCMV-CD63-L7Ae-His, expressing the fusion of the exosomal membrane proteins CD63 and L7Ae. HEK293F cells were co-transfected with these plasmids, and exosomes secreted by the cells were harvested by ultracentrifugation. The content of TH mRNA in the exosomes was detected by qPCR, and the exosomes were then transfected into recipient cells. Results: Compared to TH-Exo obtained by transfection with the single tyrosine hydroxylase plasmid, the proposed method resulted in significantly higher levels of TH RNA encapsulation in exosomes (TH-Kt-Exo). In addition, the exosomes were able to deliver the loaded mRNA to recipient cells. Conclusion: The specific binding between L7Ae and the Kt loop effectively enhances the encapsulation of the target mRNA in exosomes.
Objective: The venom glands of toxic animals secrete a large number of structurally diverse and functionally rich active peptides, representing a natural treasure trove for peptide drug development. At present, only a small portion of the active peptides from toxic animals have been studied, so there is a need to establish a more efficient method of active peptide discovery. Methods: The anticoagulant peptides were developed by transcriptome sequencing, data analysis and polypeptide selection, peptide preparation by recombinant expression, in vitro activity screening, and in vivo activity evaluation in animal models of the Draconarius digitusiformis. Results: PDBPE-001, an inhibitor of coagulation Factor Xa, was screened. The peptide has a molecular weight of 9 889.82 Da, consisting of 92 amino acid residues and 4 pairs of disulfide bonds. This peptide can be efficiently prepared through recombinant expression, and its inhibitory activity against Factor Xa is concentration dependent, with an IC50 ~ 0.807 μmol/L. In the mouse thrombus model, 30 mg/kg of PDBPE-001 has a good anti-thrombotic effect. Conclusions: A novel Factor Xa inhibitor has been obtained for the first time from the Draconarius digitusiformis, providing a new lead peptide molecule for the development of new anticoagulants.
The mature zygotic embryos of Larix gmelinii were used as explants to study the isolated organogenesis of larch. The experimental design used different medium, including 6-BA and 2,4-D to induce healing and adventitious shoots, elongation and rooting. The results showed that the best callus induction medium was 1/2 MS + 2 mg/L 2, 4-D, and callus induction rate was 91.11%. Induction medium for adventitious budding was 1/2 MS+1 mg/L 6-BA+0.07 mg/L 2, 4-D, and the adventitious shoot induction rate was 87.78%. The indefinite bud elongation medium was MS+0.20 mg/L 6-BA+0.01 mg/L 2, 4-D + 1 mg/L activated carbon, and the elongation rate of adventitious buds reached 48.33%. Adventitious root rooting medium was 1/2 MS + 1 mg/L IBA, and the induction rate of indefinite root reached 36.67%. The survival rate of transplanted seedlings was 100%. The above studies provide a good theoretical and practical basis for larch tissue culture organogenesis and further research on stable genetic transformation.
Abnormal protein expression often leads directly or indirectly to the development of cancer. Currently, small-molecule inhibitors that target specific proteins are widely used in tumor therapy. However, small-molecule inhibitors have problems such as susceptibility to drug resistance, limited range of target proteins, and high toxicity, which limit their clinical application. As a result, proteolysis-targeting chimaera technology has emerged. Proteolysis targeting chimaeras (PROTACs) are a type of synthetic small-molecule compound consisting of target protein ligand, linker and E3 ubiquitin ligase ligand. It can use the human body’s own ubiquitin-proteasome system (UPS) to ubiquitinate and degrade the target protein, which to some extent solves the problem of drug resistance caused by overuse of small-molecule inhibitors, and has the advantage of low toxicity. Therefore, this review has summarized the design and application of PROTACs developed by using existing small-molecule inhibitors in the field of cancer, based on the top five cancers with the highest incidence rate in China, with the aim of giving inspiration to new drug researchers, expanding the use of small-molecule targeted inhibitors, and breaking through the selection of drugs that are difficult to become drug sites.
Cancer is a leading cause of death. Although modern medicine has made significant progress in cancer treatment, traditional treatment methods still have limitations due to problems such as cancer heterogeneity, drug resistance, and treatment side effects. With the development of science and technology, bacterial therapy has shown great potential in the field of anti-tumor therapy. Bacteria have the natural ability to target tumors due to their survival characteristics and contain a large number of immune-activating substances, which can modulate the tumor microenvironment and activate the immune system for tumoricidal effect to kill the tumor. Some bacteria can also directly kill tumor cells and inhibit tumor angiogenesis through various pathways. Through in-depth research, scientists have also found that bacteria combined with radiotherapy, chemotherapy, and immunotherapy have gradually become the mainstream strategy of bacterial therapy. In addition, exogenous genes can be inserted into bacteria to perform specific functions according to clinical needs. When combined with various treatment modalities, it is possible to improve their therapeutic effect against tumor, and reduce the toxic side effects. The research progress of bacteria in anti-tumor therapy has been summarized from the aspects of the basic principles, types of bacteria mainly used, optimization strategies, and clinical trials and cases, in order to provide more effective therapeutic strategies for cancer patients by fully exploiting the potential of bacteria.
Cellular metabolic reprogramming plays an important role in maintaining cellular homeostasis, cell growth and proliferation, and is widely involved in pathological processes such as malignant transformation. With the development of high-throughput molecular detection technology, it has been found that some metabolic enzymes can not only participate in the regulation of cellular metabolism by catalyzing a variety of intracellular biochemical reactions, but also bind to RNA molecules. These metabolic enzymes do not have classical RNA-binding domains, and it has been shown that they can regulate the transport, stability, or translation of their binding mRNA through a negative feedback mechanism, thereby linking the regulation of gene expression to cell metabolism. In addition, the metabolites of enzymes may also be involved in the regulation of the interaction between RNA and metabolic enzymes. This study reviews metabolic enzymes with RNA-binding ability, the interaction between metabolic enzymes and RNA, the identification and verification of RNA binding protein, metabolic regulation mechanisms, and how the interaction between these metabolic enzymes and RNA regulates complex cellular activities and the onset and development of disease.
Extracellular vesicles are natural nanoparticles with a phospholipid bilayer membrane structure released by cells. They play a vital role in various physiological and pathological processes such as cell signaling, tumor development, immune regulation, and rejuvenation during aging. They have immense potential in the diagnosis and treatment of disease. Previous research has shown that the preparation of extracellular vesicles is prone to protein contamination, which limits their research and translational applications in biomarkers and drug delivery. In recent years, some scholars have introduced the concept of “protein corona” to the field of extracellular vesicles, which is proven to be an important structure for synthetic nanoparticles. They propose that the protein corona is an intrinsic component of the extracellular vesicle and significantly influences its biological functionality. This challenges the traditional view of protein contamination and leads to a paradigm shift in extracellular vesicle research. This review provides an overview of the current state of research on the protein corona on the surface of extracellular vesicles. It covers aspects such as the formation process, chemical composition, biological functions, and identification methods of this protein corona. This review may serve as a reference for the further study of extracellular vesicles and their protein corona.
In recent years, China’s cellular immunotherapy has developed rapidly, catching up from zero to the international level of excellence. Behind the booming development of cellular immunotherapy, the support of vector technology for gene delivery is indispensable. As a medium for introducing genes into target cells and enabling their expression, vector technology is also one of the major bottlenecks in the development of the industry in terms of how to carry out gene transfer safely and efficiently. By summarizing the current status of the development of vector technology for major applications in the field of cell therapy and comparing the industrialized production process of already marketed products, we hope to provide a reference for the further development of vector technology.
Bismuth (Bi), as a heavy metal of low toxicity, has been used to synthesize various nanomaterials with unique structure and physical and chemical properties. The synthesized bismuth-based nanomaterials have the characteristics of good biocompatibility, a favorably high X-ray attenuation coefficient, a long circulation half-life, high stability, excellent light-to-heat conversion efficiency and photocatalytic ability. Due to these properties, bismuth-based nanomaterials are widely used in biomedical fields such as tissue engineering, antibacterial materials and anti-cancer treatment. It is reported that bismuth-based nanomaterials have been made into drugs for the treatment of diseases. Compared with traditional antibacterial drugs, bismuth-based nanomaterials as antibacterial agents can effectively avoid the occurrence of bacterial drug resistance. The properties, antibacterial mechanism and progress in the antibacterial field of bismuth-based nanomaterials are reviewed. Furthermore, the future research direction of bismuth-based antibacterial nanomaterials is proposed.
Highly efficient bioproduction of hydrocarbons is one of the effective means to solve the shortage of petroleum and other liquid fuels, while microalgae-based oils are a reliable choice for the production of sustainable biofuels. Botryococcus braunii is an irregular unicellular colony belonging to the chlorophyta, which has received widespread attention for accumulating the large amounts of hydrocarbons, with a maximum content of up to 75% of its dry weight. In recent years, continued in-depth research into the biological characteristics and growth physiology of B.braunii, has improved the feasibility of its large-scale cultivation and industrial production of hydrocarbons. In this review, the potential of B.braunii as a novel oleaginous microalga for hydrocarbon production was briefly described from the aspects of biological characteristics, hydrocarbon synthetic pathways and regulatory factors, omics studies, and large-scale cultivation techniques. This could provide a reference for exploring the large-scale industrial production of biofuels based on B.braunii, thus accelerating the development and exploitation of this microalgal resource.
The application of gene regulatory tools in the field of cyanobacterial synthetic biology is particularly important for controlling gene expression, engineering strain development, and the production of additional economic products. Small RNA regulatory tools, which are based on the principle of targeted specificity regulation of small RNAs, are quantitative and global regulatory tools that are combined with synthetic biology for specific regulation of target genes. These tools use small RNAs and their targets as interaction modules and use inducible expression switches, scaffold sequences, and partner proteins as ancillary modules. Such tools have been applied to the synthesis of high-value economic products and the modification of algal strains for fuel and chemical tolerance. This paper provides an overview of different types of small RNA regulatory tools, their regulatory principles, the selection and modification of ancillary modules, as well as research progress in the synthesis of additional economic products and improvement of cyanobacterial tolerance. It also discusses potential problems and future development prospects of small RNA tools in applications, providing a reference for the design of efficient and precise molecular editing tools.
Targeted protein degradation (TPD) technology refers to a type of technology that utilizes the internal naturally occurring protein purification system to reduce target protein levels. By analyzing the status of basic research and industrial development status of TPD, it can be seen that the development of TPD has entered the fast lane in recent years. With the progress of research, the resources of degradable target protein and recruitable E3 ubiquitin ligase have been expanded, and the stability and efficacy of TPD have been further improved, which realizes controllable and precise degradation. At the same time, the target protein has been expanded from intracellular proteins to extracellular and membrane-binding proteins, protein aggregates, and non-protein substances such as RNA, lipids, organelles, and pathogens based on technological prototype innovation. TPD technology has also provided new ideas for the development of new drugs against undruggable targets, ushering in a new wave in the pharmaceutical industry, and opening up new avenues for the treatment of diseases such as cancer and autoimmune diseases.
