Objective: Study the effect of deubiquitinating enzyme USP13 on the proliferation and apoptosis of human chronic myeloid leukemia K562 cells, and explore the underlying mechanism. Methods: Construction of the pLKO.1-shUSP13-GFP lentiviral interference vector and establishment of the USP13 knockdown K562 cell line using lentivirus. Western blot detected the USP13 knockdown efficiency in K562 cells. Flow cytometry analyzed the effect of USP13 knockdown on the proliferation and apoptosis of K562 cells. Co-immunoprecipitation and protein ubiquitination experiments explored the regulation mechanism of USP13 on K562 cells. Results: The pLKO.1-shUSP13-GFP vector was successfully constructed, and K562 cell line with stable knockdown of USP13 was obtained using the lentiviral system. Flow cytometry results showed that knocking down USP13 promoted K562 cell apoptosis and inhibited cell proliferation. Molecular mechanism studies found that knockdown of USP13 decreases c-Myc level by enhancing its ubiquitination. Conclusions: Data preliminarily revealed the molecular mechanism of USP13 regulating the proliferation and apoptosis of K562 cells, providing a potential target for the treatment of chronic myeloid leukemia.
Aim: To investigate the effect of miR-29a on lipopolysaccharide-induced injury in human pulmonary microvascular endothelial cells and its potential mechanism. Methods: The LPS-induced HPMVECs injury model was constructed. The expression level of miR-29a was detected by RT-qPCR. The concentration of LDH was measured by ELISA. MTT assay was used to detect the cell proliferation. The flow cytometry was applied to determine the HPMVECs apoptosis. The protein levels of PTEN, p-Akt, Akt, p-FOXO3a, FOXO3a and Bim were determined by Western blot. The targeting relationship between miR-29a and PTEN was predicted by Microcosm, starBase, Pictar, TargetScan and confirmed by luciferase test. Results: LPS treatment of HPMVECs significantly reduced the expression level of miR-29a and cell viability,induced the increase of LDH release amount and cell apoptic rate, upregulated the expression of PTEN and Bim protein, and down regulated the expression of p-Akt/Akt and p-FOXO3a/FOXO3a (P<0.05). Overexpression of miR-29a reversed the injury of LPS to HPMVECs. Dual luciferase reporter gene assay confirmed that PTEN was a negative regulatory target gene of miR-29a. The protein expression of PTEN was significantly down regulated by miR-29a mimics, and up-regulated by miR-29a inhibitors(P<0.05). However, the expression level of PTEN mRNA had no statistically significant difference (P>0.05). Conclusion: Overexpression of miR-29a, which targets inhibition of PTEN protein expression, protects against LPS-induced HUVECs injury by activating the Akt/FOXO3a/Bim pathway.
Objective: To meet the requirements of rapid and efficient gene editing detection in high-throughput screening applications, it is of great significance to establish an in situ evaluation method on cells. EGFP can be used to evaluate the gene editing performance mediated by the CRISPR system, but the efficiency is limited by the long half-life of EGFP. Methods: A version of destabilized EGFP (EGFP-PEST) was constructed by fusing degradation domain of ornithine decarboxylase (containing PEST motif) to EGFP. The EGFP-PEST gene was introduced into the chromosome of HEK-293T cells by lentivirus, and the copy number of EGFP-PEST gene was measured by RT-qPCR. Finally, cell strains with single copy of EGFP-PEST transgene were established. Results: Compared to unmodified EGFP, the cellular fluorescence of EGFP-PEST significantly decreased within 4 h, suggesting efficient PEST-mediated protein degradation. The cell model was used to evaluate the potential of three commercial lipids to deliver CRISPR/Cas9 complex. Data showed that gene editing was detected in 2-4 days by quantitative or qualitative measurements. Conclusion: This cell model can be used in high-throughput screening for new CRISPR tools or novel delivery systems by indicating the gene editing rapidly and sensitively.
Objective: To establish a colloidal gold technique assay for the rapid detection of antigen against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to evaluate its clinical performance. Methods: The colloidal gold was prepared by trisodium citrate reduction. The monoclonal antibody of mouse anti-SARS-CoV-2 nucleocapsid protein (NP) and dinitrophenol-bovine serum albumin (DNP-BSA) were labeled with colloidal gold nanoparticles. The monoclonal antibody of mouse anti-nucleocapsid protein and rabbit anti DNP polyclonal antibody were coated on the nitrocellulose membrane as detection line and quality control line to prepare immunofluorescence test strip. The performance of the limit of detection, cross-reactivity, accelerated stability, sensitivity and specificity of clinical diagnosis were evaluated. Results: The limit of detection for heat inactivated SARS-CoV-2 was 2.0×102 TCID50/mL. There were no cross reaction with high concentration samples or cultured virus of 16 common pathogens. The kit was stable after 8 weeks accelerated at 50℃. Nasopharyngeal swab samples of clinical and healthy people were tested, the sensitivity was 96.67% (29/30), the specificity was 99.23% (129/130), the total coincidence rate was 98.75% (158/160), and the Kappa consistency test had a Kappa value of 0.959 0 (P<0.05). Conclusion: The SARS-CoV-2 antigen detection reagent (colloidal gold method) has the advantages of high sensitivity and specificity, fast detection speed, portable operation, no need for equipment and naked eye observation, which can be used as a supplementary method for the existing SARS-CoV-2 nucleic acid detection method.
Pectinase preparations have been widely used in China. However, efficient and specific pectinases are still lacking in the market. Producing a single-component pectinase using modified Aspergillus niger by genetic engineering technology has become an effective solution to meet the growing industrial demand of pectinase. Constructing an efficient CRISPR-Cas9 technology can provide an efficient genome editing tool for the construction of Aspergillus niger chassis strain with high yield single pectinase. First, the pyrG gene on the genome of pectinase-producing Aspergillus niger was knocked out to construct the uracil auxotrophic strain AnΔpyrG, and the Cas9 expression cassette and pyrG expression cassette were integrated precisely at the pyrG site of the AnΔpyrG strain to construct AnCas9 strain constitutively expressing the Cas9 gene. Then, the pLM2-sgRNA plasmid which contained the gpdA promoter, hammerhead ribozyme, and HDV ribozymes for the efficient expression and maturation of sgRNA was constructed. And finally, the CRISPR-Cas9 gene editing system is successfully established. The 4978020 and the 4983861 gene were used to detect the gene editing efficiency of the constructed CRISPR-Cas9 system, and the phenotypic changes and enzyme production changes of the 4978020 gene function deletion strain were detected. The results showed that the pectinase gene editing efficiency is more than 50%, the phenotype and pectinase activity of the strain AnΔ4978020 had no significant changes compared with the original strain. An efficiency CRISPR-Cas9 system was successfully constructed in Aspergillus niger producing pectinase, and losing of the 4978020 gene function did not affect the phenotype of the strain, which laid the foundation for the construction of high yield single pectinase Aspergillus niger chassis strain.
PiRNA is a member of non-coding small RNA, which is commonly detected in germline cells. Previously scholars used to believe that piRNA mainly plays a role in maintaining the function of stem cells,formation of the gametes and silencing foreign transposon genes.But recent discoveries which show the piRNA traces in somatic cells have led to greater interest in its biogenesis and further function. The discovery, structure, function and gene regulation of piRNA were reviewed.
Terminal deoxynucleotidyl transferase (TdT) is a member of the polymerase X family. Unlike typical DNA polymerases, TdT incorporates nucleotides at the 3'-end of single stranded DNA oligo in a unique template-free manner. Moreover, the high tolerance of TdT to substrates allows it to polymerize modified dNTP. Fluorescence-modified dNTPs, biotin-modified dNTPs and even artificial bases can be used as good substrates. These biochemical properties of TdT make it widely used in the fields of biosensing and nucleic acid synthesis, promote the development of many nucleic acid-based tools and methods, and lay the foundation for the development of enzymatic de novo DNA synthesis technology. The latest progress of cartilage 3D bioprinting and the limitations of current technology are also explained.
Self-repair is critical but limited on aged and damaged articular cartilages, and therefore, it is necessary to apply a suitable tissue engineering scaffold to promoting the repair and growth of the defective cartilage. 3D bioprinting can accurately distribute cell-bearing biological materials and construct complex three-dimensional living tissues or organs, and such technique becomes a hotspot in cartilage tissue engineering recently. The focus is on the latest progress of cartilage bioprinting in 3D bioprinting, including the selection of “ink” materials for cartilage bioprinting, the source of seed cells, and the development of 3D bioprinting technology. In addition, some limitations of the application of 3D bioprinting technology in tissue engineering are also explained, and its development and application in the field of cartilage repair are predicted.
Autophagy is a very conservative biological process of lysosomal degradation in eukaryotic evolution, which plays an important role in maintaining cell homeostasis and eliminating harmful components. Receptor tyrosine kinases (RTKs) are a class of kinase proteins that play an important role in the movement and invasion of normal cells and cancer cells. RTKs protein can not only promote autophagy, but also inhibit autophagy. Studies have shown that RTKs can play a regulatory role in tumors and related diseases through autophagy. For example, epidermal growth factor receptor (EGFR) can inhibit autophagy, thereby promoting tumor growth and proliferation; it can also pass RTK/Ras/ERK signaling pathways to induce autophagy, which in turn participates in related diseases such as cellular immune responses. The regulatory effects of RTKs on autophagy and related research results, which provide a basis for the theoretical basis of target targeted therapy were reviewed.
Exosomes are nano-vesicles secreted by cells with diameter ranging from 30 to 150 nm. Attributed to superior biocompatibility, excellent loading capacity and membrane ease to be functionalized, exosomes are considered to be a promising drug delivery carrier. Among the study of tumor therapy, exosomes with targeted recognition can be used to reduce off-target effects so as to enhance the therapeutic effect. The research progresses of different modification methods to enhance the targeting ability of exosomes were concluded, the recent research using exosomes as specific drug delivery carrier to target tumor cells was summarized, and the advantages and disadvantages of exosomes as rising drug delivery carrier, which provide feasible directions and strategies for the designing of exosomes equipped with targeting specific ability were elaborated.
Escherichia coli has become a commonly used system for expressing recombinant proteins due to its obvious advantages. However, E. coli does not have the oxidative conditions and molecular mechanisms for the formation of disulfide bonds in the cytoplasm, and high-level expression often tends to aggregate to form inclusion bodies, which limits its use. Signal peptides to guide proteins secretion can overcome this shortcoming. The proteins with signal peptides are secreted by the Sec or/and Tat systems in E. coli. The E. coli secretion systems and the structure of signal peptides were summarized. Meanwhile, the research and application progress of six commonly used signal peptides in recent years were introduced. Finally, the problems and improvement measures in the application of signal peptides were summarized,which would provide more useful information and strategies for researchers to select signal peptides reasonably and optimize the expression of recombinant proteins.
Acteoside is a natural product widely found in many plants, which is composed of caffeic acid, hydroxytyrosol, glucose and rhamnose, including anti-inflammatory, antibacterial, antiviral, anti-tumor, antioxidant, analgesic, neuroprotective, improving sexual function, immunomodulatory, and memory protective activities. However, traditional extraction of acteoside and its derivatives from plants have some problems such as low content, low extraction efficiency and environmental pollution.In recent years, the study of heterologous synthesis of plant natural products has been successfully constructed by the rapid development of synthetic biology, which provides a new idea for the efficient production of acteoside. The recent advances of acteoside cell factory which includes the key metabolic biosynthesis elucidations of phenylethanoid glycoside , mining and optimization of key gene components, synthesis of key precursors were summarized.
Itaconic acid, a platform chemical, can be widely applied in various industries for kinds of high value-added products. It is one of the potential alternatives to the traditional petroleum-based material, which has an important position and application prospect in industrial production. At present, itaconic acid is mainly produced by submerged fermentation by Aspergillus terreus. A number of parameters have significant influence on the yield of itaconic acid, such as carbon source, nitrogen source, phosphate, metal ion, dissolved oxygen, pH, and temperature. The high cost of raw material is a crucial factor which impedes the expansion and development of the itaconic acid market in industrial production. Lignocellulose is a widely available and a promising low-cost substrate. The research of lignocellulosic hydrolysate as a substitute carbon source to produce itaconic acid is expected to reduce the production cost, which is of great significance to promote the development and application of itaconic acid.
