To screen for differentially lncRNAs expression profile and predict their functional roles in the macrophage after Mycobacterium tuberculosis (M.tb) stimulation. Firstly, microarray and bioinformatics analysis of lncRNA and mRNA expression profiles in RAW264.7 macrophage after stimulation with M.tb for 24h.Then, 16 differentially expressed lncRNAs from microarray analysis were further verified by RT-qPCR using H37Rv infected macrophages and mouse model. The results shown that the expression levels of 4 730 lncRNAs was up-regulated, and the expression levels of 9 558 lncRNAs was down-regulated. 16 differentially expressed lncRNAs from microarray screening were associated with protein coding genes in adjacent locations. The mRNA function annotation analysis revealed that the mRNAs of differential expression were mainly concentrated in the biochemical process of transcriptional regulation, phosphorylation, apoptosis and MAPK signaling pathway which participating in the biochemical process of anti-tuberculosis. The expression trend of 4 lncRNAs in the iH37Rv stimulated RAW264.7 and mouse infection model was vertified as the same in both microarray and RT-qPCR analysis. Three of these 4 lncRNAs was up-regulated and one of them was down-regulated.The abnormal expression of lncRNAs may provide clues to the dysfunction of macrophages with M.tb infection, and further research will focus on the investigation of the function and regulation mechanism of lncRNA in M.tb infected macrophage.
Objective:To investigate the effects of bone morphogenetic protein 9 (BMP9) on the proliferation and migration of human bladder cancer BIU-87 cells.Methods:BIU-87 cells were infected with BMP9 recombinant adenovirus(AdBMP9); The mRNA levels of BMP9 were detected by Q-PCR; The BMP9 protein and its downstream associated signaling pathway protein levels were detected by Western blot; The MTT and colony formation assay were used to test the proliferation of BIU-87 cells; The wound healing and Transwell TM chamber assay were taken to evaluate the migration of BIU-87 cells. Results:The expression of BMP9 in BIU-87 cells was highly increased after AdBMP9 infection; Forced expression of BMP9 promotes both the proliferation and migration of BIU-87 cells; Western blot results showed that BMP9 overexpression also increased AKT phosphorylation.Conclusion:High levels of BMP9 may promote the proliferation and migration of bladder cancer BIU-87 cells by activating the AKT signaling pathway.
A new type of neuroendocrine peptide, pituitary adenylate cyclase activating peptide (PACAP), has been found to play an important role in carbohydrate or lipid metabolism but is susceptible to dipeptidyl peptidase IV degradation. Chitosan-modified nano-selenium (SeNPs-CTS, SC) was used as a carrier and the PACAP-derived peptide MPL-2 was loaded by amide bond to prepare a stable and stable nanoparticle peptide SeNPs- CTS-MPL-2 (SCM). The experimental results show that the high stability nano-composite peptide SCM was successfully constructed. The average particle diameter of SCM was 158nm, the particle size was relatively concentrated, and the surface Zeta potential was 35.6mV, which was significantly different from the SC particle size and Zeta potential. These proved that MPL-2 successfully connected to the SC surface. SCM was stable in aqueous solution for 40 days and has strong stability in aqueous solution. In vitro sustained release experiments showed that SCM released MPL-2 continuously within 48 hours, effectively prolonging the action time of MPL-2. Type 2 diabetes model mice (db/db mice) were injected intraperitoneally with SCM. Glucose tolerance test results showed that SCM sustained release of MPL-2 in vivo after MPL-2 was loaded onto carrier SC, prolonged the effect time of MPL-2 and enhanced the efficacy of MPL-2. During 8 weeks of continuous medication, SCM significantly increased insulin sensitivity in mice with type 2 diabetes mellitus, significantly more potent than MPL-2 and SC alone. The nanocomposite peptide SCM was constructed to effectively prolong the action time of MPL-2 and to exert the biological effect of treating type II diabetes.
To investigate the function of the key residues in the binding site of human uMtCK onto substrate creatine (Cr) and ATP, nine mutants of human uMtCK were constructed by using site-directed mutagenesis and analyzed by enzyme kinetics, circular dichroism spectra and protein structural simulation. The
Objective:To study the effect of shPLCε on Warburg effect in bladder cancer and the potential mechanism.Methods:(1) Using lentivirus infecting T24 cells,the glucose consumption and lactate production were detected by the corresponding kit. The expression of PLCε、CDC25A and the molecules related with Warburg effect such as PKM2, GLUT1, LDHA were detected by q-PCR and Western blot. (2)To investigate the related molecular mechanism, using plasimid infecting T24 cells, the expression of CDC25A was detected by q-PCR and Western blot. The expression of molecules related with Warburg effect were detected by Western blot.Result:(1)After reducing the expression of PLCε, glucose consumption and lactate production were significantly decreased in the LV-sh PLCε group,compared with the blank control group and LV-NC group (P<0.01).q-PCR and Western blot showed that the expression of CDC25A, PKM2, GLUT1, LDHA were remarkably lower in LV-shPLCε group than that in blank group and LV-NC group (P<0.05). (2)Western blot showed that the expression of PKM2, GLUT1, LDHA were remarkably lower in shCDC25A group than in blank group and shNC group(P<0.05), after treated with shCDC25A-plasimid.Conclusion:shPLCε can reduce Warburg effect through decreasing the expression of CDC25A in bladder cancer, which can not provide energy for the development and progression of bladder cancer.
Objective:To integrate substantial but scattered state-of-the-art precision medicine knowledge and form a systematic knowledge network, to support clinical application of individual omics data, aiming at precision medication recommendations.Methods:The database was constructed using MySQL. Precision medicine knowledge from FDA companion diagnosis, NCCN guidelines, My Cancer Genome and GDSC was manually collected in a unified format after being standardized and structured.Results:The tumor precision medicine knowledge base (PMKB) was successfully designed and constructed and has already collected 1 940 clinical directives, covering 14 kinds of variations.Conclusion:PMKB collects information relating tumor mutations and therapeutic strategies, which can provide personalized treatments of reference. PMKB is also the base of constructing a clinical decision support system of precision medicine.
Objective:To investigate the new gene Rpfan37, a homolog of PITP, in order to provide an idea for understanding the function of relative genes that involved in the symbiotic nodulation process.Methods:Through the previous study, target genes which were suspectly associated with symbiotic nodulation were screened out from the suppression substractive hybridization library of Robinia pseudoacacia interacting with symbiotic rhizobia. Using qRT-PCR to analyze the target gene expression level at different times and in different tissues. The RNAi recombinant plasmid was transformed into the plant root through K599 and verify the function of Rpfan37 after inoculation.Results:qRT-PCR analysis showed up-regulation characteristics of Rpfan37 in roots especially at 15 days post-inoculation (dpi), however, inoculation and non-inoculation treatment had no significant effect on the gene expression which sharply decreased in the matured nodule. Knockdown of Rpfan37 via RNA interference resulted in impaired development of both plant growth and nodule. Compared with empty vector plants, fresh weight, the root and stem length, nodule number per plant deceased dramatically in Rpfan37 RNAi plants. The root hair development of RNAi plant was abnormal and the number of root hair curling, ITs and nodule primordia were also significantly reduced in the Rpfan37 RNAi roots. Nodulation paraffin sections showed that the number of infected cells in RNAi plant nodules was significantly reduced compared with the control. Real-time PCR analysis of the expression levels of leghemoglobin gene indicated that nodule development and maturation was significantly blocked in the Rpfan37 RNAi roots.Conclusion:The related gene Rpfan37 found in Robinia pseudoacacia can participate in the symbiotic nodulation process. It provides a new theoretical basis for understanding the function of phosphatidylinositol transporter in symbiotic nodulation process.
Objective:Study the effect of R122 residue mutation on the stability of recombinant porcine trypsin (RPT).Methods:RPT,mutants mRPT(R122H) and mRPT(R122H/R73G/R130) were expressed in Pichia pastoris GS115 and further purified. The properties and stabilities of RPT and two mutants was investigated and compared.Results:RPT and its mutants were highly expressed in Pichia pastoris. Relative to RPT, mutant mRPT (R122H) and mRPT (R122H/R73G/R130T) showed higher affinity to substrate BAEE, The Km values were 18.8μmol/L, 9.0μmol/L and 11.0μmol/L, respectively. Increased stability of mutants to high temperature and alkali were observed. And higher resistance against autolysis were got in the presence and without Ca 2+. Conclusion:Pichia pastoris can be used to efficiently express RPT and its mutants.Increased stability under alkaline condition and higher thermal stability and higher anti-self-digestion were got in mutant mRPT (R122H) and mRPT (R122H/R73G/R130T) compared to wild-type RPT, which contribute to the site mutation at the R122.
Objective:To construct Qβ phage virus-like particles (VLPs) vaccines presenting human interleukin-13 (IL-13) antigen peptide.Methods:The human IL-13 antigen peptide was genetically recombined into the C terminus of the Qβ phage capsid protein (CP). In BL21 bacteria, the native CP and the recombinant CP with C terminal fused with IL-13 antigen peptide (CP / IL-13) were induced simultaneously by IPTG. VLPs were purified by ammonium sulfate precipitation and sucrose density gradient centrifugation, and the presence of chimeric VLPs was analyzed. The purity of VLPs was analyzed by HPLC and the morphology of the particles was observed by electron microscopy. The mice were subcutaneously immunized with VLPs and sera were collected for detection of human IL-13-specific IgG antibodies by ELISA. RESULTS: The recombinant protein CP and CP / IL-13 were successfully expressed. Both of them appeared in the same fractions of collected samples from density gradient centrifugation and had the same sedimentation behavior as the native Qβ VLPs, while CP / IL-13 alone had no Qβ particle behavior. After purification, high purity particles were obtained. The chimeric particles were similar in shape to Qβ particles. In addition, the VLPs vaccine induced mice to develop an IL-13-specific antibody response.Conclusion:The co-expression strategy can successfully construct chimeric VLPs presenting human IL-13 epitopes, and provide a vaccine form with potentials for clinical application antagonizing the pathological effects of IL-13 in severe human diseases.
Objective:Epidermal growth factor receptor (EGFR) is overexpressed in tumors and is associated with metastasis, invasion and poor prognosis of tumor cells. Therefore, EGFR has become an attractive target for the treatment of cancer. Several small molecule compounds that specifically inhibit EGFR have been clinically developed. Preparation of specific antibody to human EGFR with an identified biological activity is essential for this target-related immunotherapeutic study, such as a construction of single chain antibody fragment (scFv).Methods:Total RNA was extracted from the hybridoma cell line specific to EGFR. The light and heavy chain variable region genes VL and VH were obtained by 5' RACE technology. The constructed scFv antibody gene was cloned into the eukaryotic expression vector pcDNA3.1. ELISA was used to detect the specificity of expressed single chain antibody to human EGFR protein. The affinity between antigen and antibody was detected by Fortebio. Flow cytometry was used to detect the functional activity of single chain antibody by which binding to natural EGFR on lung cancer cell lines.Results:The VL and VH sequences of light and heavy chain variable regions were obtained, and assembled EGFR-scFv was successfully expressed. The EGFR-scFv was specifically bound to natural EGFR protein with an affinity of 3.22×10 -9mol/L, and the scFv was further indentified based on its binding to EGFR that expressed by lung cancer cell line H1975. Conclusion:The anti-human EGFR single chain antibody was successfully constructed, which lays the foundation for development of EGFR- targeted immunotherapy.
Chitosan is a biocompatible, low-toxic and biodegradable amino polysaccharide obtained by deacetylation of chitin. The classic gelling system of chitosan/β-sodium glycerophosphate thermosensitive hydrogel has been widely reported in tissue engineering, drug controlled release and other fields. The gels properties depend on the composition and concentration of solutions. To improve the defects of weak mechanical property, rapid degradation and drug burst release, researchers modified chitosan or blended it with other materials, expecting to get better thermosensitive hydrogels based on chitosan. The recent advances in chitosan-based thermosensitive hydrogels including modified chitosan and blend hydrogel are summarized. Also summarizes the applications of those hydrogel in tissue engineering (repair of cartilage, blood vessels and nerve) and drug delivery release (controlled release of cancer drugs, diabetes treatment), in order to provide a reference for further research on the thermosensitive hydrogels.
Lignocellulosic biomass, a kind of renewable and widely distributed resource on earth, serves as a potential source for the production of bio-based energy, bio-based materials and bio-based chemicals. Resistance barrier is the major limitation in conversion of lignocellulosic biomass. The main components of lignocellulosic biomass are cellulose, hemicellulose and lignin. Lignin interacts with hemicellulose through hydrogen or covalent bonding, acting as a physical barrier that restricts the access of cellulose. Furthermore, there is a strong hydrogen bonding among crystalline cellulose. Though several pretreatments are available, biological pretreatment seems to be promising as an eco-friendly process and has been paid much attention by many researchers. The effects of biological pretreatment on cellulose, hemicellulose and lignin were evaluated. The degradation and modification of lignin, variation of cellulose content and crystalline cellulose, production and utilization of pentose from hemicelluloses, and microstructure changes during the biological pretreatment were systematically reviewed. In addition, the future prospect of combination pretreatments, multi-enzyme catalytic system, biomass component fractionation and utilization, and the efficient bacterial pretreatment were put forward.
Disruptive technology is based on the new technology development track, it has the characteristics of initial phase such as low-end and marginality, and will become the mainstream technology. The article designed a method to identify disruptive technology based on multi-data sources to character the different link of human innovation and knowledge relationship between them. Then it research on the evolution process of scientific knowledge, on tracing the evolution trail of advanced technologies through the knowledge relationship of multi-data sources which including data generation, development, citation and reproduction. In order to discover and identify the disruptive technical themes. The scientific and application status of cellulose biodegradation technologies are analyzed, such as strain breeding, enzyme engineering, fermentation technology, separation and purification etc., through mining multi-data sources about fund programs, conferences, fundamental and application research information, patents, business reports. Finally, the evolutionary trend of cellulose biodegradation, calculated the happen time of disruptive technologies of cellulose biodegradation, and drew the industrial technology roadmap are analyzed.
Immunotherapy has been successfully applied in the treatment of many kinds of tumors, and significantly improved the quality of life of patients. Immune cell therapy is one of the focus directions in the development of immunotherapy. The development of immune cell therapy has gone through the stage of development of non-specific immunity to non-differentiated specific immunity and to differentiated specific immunity. Several immune cell therapy products have launched, and the industrial system has been preliminarily formed, including treatment/drug development and related services/equipment production. By analyzing the immune cell therapy industry development situation at home and abroad and the technology development bottleneck, main problems of the development of the industry in China are pointed, some suggestions were put forward so as to provide reference for the development of immune cell therapy industry in China.
