The present study investigates the effect of JNK pathway on the polarization of M2 status as well as pro-tumor function mediated by M2. THP1 derived M2 macrophage (THP1-M2) model was established. The cells were divided into 3 groups: the PMA pretreated unpolarized macrophage (M0), the PMA and IL-4 induced M2 macrophage with DMSO (negative control) treated (M2), the JNK inhibitor treated M2 macrophage (M2-JNK I). Furthermore, the M2 associated markers Arginase1 (Arg1),mannose receptor C-type 1 (Mrc1)were analyzed by Q-PCR, the protein level of Arg1 and Mrc1 were detected by Western blot, the migration ability of macrophages was tested by Wound Healing, the apoptosis of 786O and OSRC2 were analyzed by flow cytometry. Compared with the THP1-M2, THP1-M2-JNK I group showed decreased expression of Arg1 and Mrc1, and migration ability was impaired. What’s more, block of JNK pathway inhibited the pro-tumor function of M2 on 786O and OSCR2. Taken together, the results suggest that inhibition of JNK pathway regulates M2 polarization and its pro-tumor effects.
Plant cysteine protease inhibitors play an important role in defense of biotic and abiotic stresses. The expression of the AtCYSa gene in Arabidopsis thaliana can be induced by a variety of stresses, and the over-expression of AtCYSa in Arabidopsis thaliana can enhance the ability to resist abiotic stress like salt, drought and oxidation. In order to further explore the function of AtCYSa and its application in tobacco, the plant expression vector pCAMBIA 1302-AtCYSa was constructed. Four positive transgenic tobacco lines were obtained by PCR and RT-PCR confirmation. Three transgenic tobacco lines were selected for salt stress treatment and insect resistance. In the salt stress treatment, the content of malondialdehyde in 100mmol/L NaCl and 200mmol/L NaCl treatment group was significantly lower than that in wild type. Evans blue staining and cell relative activity results showed that the cell activity of transgenic tobacco was significantly higher than that of wild type. This indicated that the expression of AtCYSa gene could protect the transgenic tobacco under salt stress. Insect activity studies showed that the total weight of the larvae in the experimental group havig a significant decrease, and the larvae mortality was significantly higher than the control. These results indicate that over-expression of the AtCYSa gene in tobacco can enhance the salt tolerance and insect resistance of transgenic tobacco.
To investigate the antitumor mechanism of EM-12, an ethanol extracts from Elephantopus mollis H.B.K., in ovarian cancer.
The effects of EM-12 on the cell viability of 2774-C10 and L02 cells were detected by MTT assay, and colony formation assay. Gene ontology analysis plots for EM-12 regulated gene, as determined by RNA-Seq. Cell cycle was evaluated by PI single staining. Cell apoptosis was evaluated by Annexin V FITC/PI staining. Expressions of apoptosis-relative proteins,and cell cycle-relative in 2774-C10 cells were measured by Western blotting.
The cell viability of 2774-C10 was inhibited by EM-12 in a dose manner. Colony formation assays showed that EM-12 decreased colony formation compared with control. Flow cytometry analysis showed an increase of the percentage of apoptotic cells and G1/S phase in a dose-dependent manner treated with EM-12. Gene ontology analysis result showed that the gene set related to G1/S cell cycle arrest scores was enriched in EM-12 treated 2774-C10 cell. EM-12 downregulated the expression of cyclin E2, cyclinD1, CDK2, and CDK6 with different concentrations. In addition, active bands of caspase-3 and PARP could be detected using Western blotting.
EM-12 induces the G1/S cell cycle arrest. In addition, EM-12 induces apoptosis by death receptor pathway.
NAC transcription factors are one of the largest plant-specific transcription factor families, and play important roles in plant growth and development, such as biotic and abiotic stress response, hormone signaling pathway, plant secondary growth, cell division and plant senescence. The CiNAC1 transgenic Arabidopsis thaliana homozygous lines was compared with wild type for further functional analyzes. The results showed that CiNAC1 overexpression lines promoted leaf senescence after ethylene treatment,furthermore, the chlorophyll contents of leaves were remarkably lower in transgenic lines than in wild-type, and ion leakage increased in transgenic lines. Quantitative real time PCR analysis showed that the expression level of chlorophyll catabolic genes, SGR1, SGR2 and PPH as well as senescence related genes, such as SAG13, SAG29, ORE1, SINA1,VNI2 and EIN3, a key transcription factor in ethylene signaling, was significantly higher in transgenic lines than in wild-type under ethylene treayment. Taken together, these results indicate that CiNAC1 play a role in ethylene induced leaf senescence in A. thaliana.
White spot syndrome virus (WSSV) is one of the most harmful viruses in the shrimp industry, and there has been no effective drug on mass scale up to now. In recent years, the immunological prevention of WSSV from shrimp has been progressed hopefully.Vp28 protein is the major structural component on the envelope of WSSV. Since 2004, its coding genes have been expressed in 8 species, and the control of WSSV has been proved remarkably in the laboratory. However, it has not been applied in shrimp industry yet. The shrimp bait, Synechococcus sp.PCC7942 was used as the acceptor to express vp28 gene, and this homology of medicine and food may be helpful for its application in the shrimp industry. The expression efficiency of vp28 in transgenic Synechococcus sp.PCC7942 has been detected by RT-qPCR method. And photosynthetic characteristics of transgenic Synechococcus sp.PCC7942 at different temperature, illumination, pH and salinity have been measured by the method of oxygen electrode. The expression efficiency of vp28 gene was 9.52% which was three times higher than that of Anabaena sp. PCC 7120. The most suitable harvest time was in late logarithm growth (the 15 th d). The optimum growth conditions of transgenic Synechococcus sp.PCC7942 were as follows: the temperature 40℃, the salinity 0~0.1mol/L NaCl, pH 7.5, light intensity 450μmol/(m 2·s). These data may be useful for scale preparation of oral drug made from transgenic Synechococcus sp.PCC7942.
Cellobiose can effectively induce the production of cellulase by filamentous fungi. Our previous studies showed that Rhizopus stolonifer TP-02 has a cellobiose synthase (CBS) that can synthesize cellobiose by utilizing uridine diphosphate glucose (UDPG) as the glycosyl donor, thereby opening the self-induced synthesis pathway of cellulase from glucose. To study the intracellular biosynthesis pathway of cellobiose, the pyrithiamine resistance gene ptrA was inserted into the GDP-glucose pyrophosphorylase gene ggp by overlapping PCR. The fused gene ggp-ptrA was respectively transformed into TP-02 and △ugp for constructing the △ggp and △ugp/△ggp mutants. LC-MS was used to analyze the intracellular sugar components of mutants. The results showed that the lack of ggp has a weak effect on the synthesis of intracellular cellobiose, while the lack of ugp directly inhibits the synthesis of disaccharides. The result of RT-qPCR showed that the transcription level of cellulase genes in △ggp mutant are 20% lower than that of the original strain, while the tested gene in △ugp/△ggp are down-regulated by 80%. Furthermore, the expression levels of cellulase were also studied. However, the FPA activity of △ugp/△ggp was not detected. These results showed that UDPG is the major glycosyl donor for intracellular synthesis of cellobiose in R. stolonifer, whereas GDPG may be the substitute for UDPG, maintaining the synthesis of disaccharides in the absence of UDPG. In addition, bioinformatics methods were used to analyze the structure and function of CBS. Through alanine scanning the Asp210 and Asp300 were confirmed as the key residues of CBS to synthesize the cellobiose, providing a direction and theoretical basis for further research and rational transformation.
According to the different N-terminal positive charges, Sec and Tat passway signal peptides were selected to construct the shuttle plasmid with pMA5, which was the first time that the leucine dehydrogenase gene from Bacillus cereus was efficiently secreted in Bacillus sutilis. The results showed that PhoD signal peptide had the highest exogenous activity, reaching 20.25U/ml, which was 2.2 times higher than that without signal peptide. The enzymatic properties of the purified leucine dehydrogenase were investigated. The purified enzyme expression level was 13U/mg; the Michaelis constants Km and Vmax were 6.17mmol/L and 14.49μmol/(L·min) for L-Leucine, separately. The enzyme had the best affinity with the natural substrate L-Leucine, and showed activity toward some the aliphatic amino acids; no activity was observed for the Aromatic amino acids L-Phenylalanine; the optimum pH was 10.5-12.0 and the stability was maintained at pH 5.0-11.0; the optimum reaction temperaturewas 55℃, The changed of the secondary structure at different temperatures by circular dichroism proved that the α-helical t α-helix content gradually decreased with increasing temperature; DSC measured Tm value of 64.13℃,indicating that the enzyme had high thermal stability.
To develop monoclonal antibodies (mAbs) against tree shrews CD3ε, and identify their biological characteristics.
BALB/c mice were immunized with GST-CD3ε protein as an immunogen. Immunized mice spleen cells were fused with SP2/0 cells with the hybridoma technique. GST- CD3ε proteins and GST proteins were used as coating antigens to establish the indirect ELISA. After screening and three rounds of cloning process, the strain of hybridomas secreting anti-CD3ε mAbs were obtained. More anti- CD3ε mAbs were prepared by mice ascites, which were purified by Protein A resin. Then anti- CD3ε mAbs were identified with indirect ELISA, superposable ELISA, antibody titer evaluation, Western blot and FACS.
Five hybridomas were obtained, and named 78I, 87I, 92D1, 75II and 35C8.The titer of five ascites were 1:10 6, 1:10 6, 1:10 4, 1:10 6 and 1:10 3. The dissociation constant (Kd) of these ascites were 1.8×10 -5, 2.9×10 -5, 4.9×10 -5, 7.3×10 -5 and 3.6×10 -5. Monoclonal antibody epitope analysis revealed that 78I, 87I, and 75II recognized the same antigenic epitope, whereas 92D1 and 35C8 recognized another antigenic epitope of GST- CD3ε. Western blot analysis showed that HRP-labeled 92D1 recognized GST-CD3ε and tree shrews’ PBMC, and had an antibody cross-reactivity with PBMC from rats, mice and monkeys. After FACS detection, 92D1 labeled with PE-Cy5.5 can specifically identify the PBMC of tree shrews.
Murine anti-tree shrews’ CD3ε mAbs were successfully prepared, which laid the foundation for the further application in immune detection of tree shrews.
Acyl-acyl carrier proteins (acyl-ACPs) are substrates for many biosynthesis pathways. However, acyl-ACP is substituted by acyl-CoA for studies in vitro as a result of supply restriction, which causes many questions in enzymatic analysis. Thus, obtaining large scale of acyl-ACP steadily is very important to study the related enzymes and metabolic pathways in vitro. acyl-ACP synthetase catalyze the conversion of holo-ACP using fatty acid as acyl donor in vitro while no productivity has been reported before. Here an acyl-ACP synthetase from Vibrio harveyi was used to catalyze the synthesis of (C4~C18) with holo-ACP, and the yield of acyl-ACP was confirmed by high performance liquid chromatography (HPLC). The results indicated that the yields of medium chains (C4~C14) acyl-ACPs were more than 90.0% while the long chain yields of 16:0-ACP and 18:1-ACP were 85.9% and 25.7%, respectively. Via introducing Li + to the reaction system, the yield of long chain acyl-ACPs were elevated above 90.0%。Then the reaction parameters were optimized in the enlarged reaction system, and more than 20mg acyl-ACPs were steadily obtained. Additionally, two species of holo-ACP were used to validate the versatility of the reaction system. Finally the acyl-ACP activity was conformed using a glycerol-3-phosphate acyltransferase. The synthesis of different chain acyl-ACPs are of great significance for research of the catalysis mechanism of related enzymes.
To prepare a carrier for NGF with injectable chitosan-hyaluronic acid hydrogel,and studying its physicochemical properties and biocompatibility.
Preparing chitosan-hyaluronic acid hydrogel crosslinked with genipin, inverted method was used to determine the gelation time; scanning electron microscopy was used to observe the morphological structure; the physical and chemical properties were measured by NGF release, vitro swelling and degradation assay; the biological properties were measured by MTT, NGF activity assay and Co-Culture experiment.
Injectable hydrogel which had a structure porous network congealed at 37℃ in about 37 minutes, and its mostly degradation degree in 8 weeks was 76%, sustained release for 21 days with biological activity. Moreover, the chitosan-hyaluronic acid hydrogel could promote RSC96 adhesion, proliferation, migration and release of cell active substances.
Genipin-crosslinked chitosan-hyaluronic acid hydrogel have good biocompatibility, and can be used as a carrier of NGF. It could be used as a potential filling material for nerve conduits.
The antigen-antibody specific binding reaction is widely used in immunoassays and biochips in which antibody immobilization play a key role in the development of efficient diagnostics and separation tools. Progress in the field of biomolecular engineering, material chemistry and crosslinker chemistry have greatly promoted the development of antibody immobilization techniques. Antibody can be immobilized on different types of solid-phase surfaces by physical adsorption, covalent attachment and affinity-based interaction. The aim of antibody immobilization is to full retain antibody comformation and activity while maximize the antigen binding capacities by immobilizing antibody to the surface in the right orientation which is critical to the analytical performance. The most recent methods for immobilization of antibody on solid-phase surface, including physical adsorption, covalent binding through carboxyl, amine, thiol, carbohydrates as well as click chemistries, and through bioaffinity techniques. The characterization methods for the investigation of immobilized antibodies are summarized. In addition, future perspectives for methods of antibody immobilization are also discussed.
With the progress of molecular biology research, the influence of various biological characteristics of the organism seeks the reason at the molecular level. Transcription factors as the important protein regulate gene transcription, and they have been explore the types and functional identification. KLF8 (Krüppel-like factor 8) is a transcription factor belonging to the family of Krüppel-like factors (KLFs), which plays a significant role in cell invasion and epithelial-mesenchymal transition, cell carcinogenesis and tumor, cell cycle and adiposity differentiation. Due to its various biological functions, it gradually becomes a hot spot for researchers to explore. The structure and function of this transcription factor has been well understood. The research progress on the molecular structure and biological characteristics of KLF8 gene and protein are explained. The gene can be used as a genetic marker and provide a reference for related research such as cancer regulation, obesity treatment and basic research.
To analyze the development status and trend of Oligonucleotide therapies in the sense of product manufacturing.
Based on the Cortellis database of Clarivate Analytics, this paper analyzed the searching results with utilizing quantitative analysis and comparative analysis methods.
Currently, 7 Oligonucleotide therapies have been launched into markets, another 4 Oligonucleotide therapies are at registration phase and 16 at Phase III clinical trial. In addition, business deals related to Oligonucleotide therapies are increasing in recent years, including more than 12 deals so far, ranging from drug development, commercial license, and patent assets sales to drug R&D cooperation in early phase. As for China, several Oligonucleotide therapies are at clinical stage, and a bright prospect can be expected for Chinese Oligonucleotide therapeutic product markets.
Although Oligonucleotide therapies market still at its preliminary stage, however, with the continuous development and improvement of future technology, it is believed that more Oligonucleotide therapies will be launched in the future, providing a new opportunity for the treatment of cancer and other diseases.
Immunotherapy based on chimeric antigen receptor (CAR)-engineered T cells has been a newly efficacious treatment for malignant tumor. The information on the development trend of domestic and global patent applications, major patent applicants and patent application by regions are acquired by searching and analyzing the patent application documents with respect to CAR-T. Particularly, the R&D technical roadmap of Novartis which received first ever FDA approval for a CAR-T cell therapy and the patent application layout of major domestic research institutions are summarized.
