Applying WCACG modified process is beneficial on reduced door-to-balloon time of acute STEMI patients.

BACKGROUND: Various systems have employed with the objective to reduce the time from emergency medical services contact to balloon inflammation for ST-elevation myocardial infraction (STEMI) patients. The WCACG message system was used to an alternative communication platform to improve confirmation of the diagnosis and movement to treatment, resulted in shorten the door-to-balloon (D-to-B) time for STEMI patients. METHODS: We collected 366 STEMI patients admitted at the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Department of Cardiology, during the period from June 2013 to October 2015. The patients were divided into two groups one underwent the current GC processes and the other group was handled using WCACG system. We compared between two groups with several indicators including D-to-B time, duration of hospitalization, associated costs, and incidence of adverse cardiovascular events. RESULTS: The results show that the new method with WCACG system significantly reduced the average D-to-B time (from 100.42 ± 25.14 mins to 79.81 ± 20.51 mins, P < 0.05) compared to the GC processes, and also reduced the duration, costs and undesirable cardiac incidence during hospitalization. CONCLUSIONS: The modified WCACG process is an applicable system to save pieces of time and efficiently integrate the opinions of experts in emergency.

Dilation catheter-guided mini-forceps biopsy improves the diagnostic accuracy of malignant biliary strictures.

BACKGROUND: Tissue sampling for biliary stricture is important for differential diagnosis and further treatment. The aim of this study was to assess a novel dilation catheter-guided mini-forceps biopsy (DCMB) method in the diagnosis of malignant biliary strictures. METHODS: 42 patients with malignant biliary stricture who underwent both brush cytology and DCMB during endoscopic retrograde cholangiopancreatography between October 2014 and November 2015 were retrospectively included. During DCMB, the mini biopsy forceps was introduced into the biliary stricture through the dilation catheter, and then the position and direction of the forceps were adjusted to obtain tissue samples. RESULTS: The positive rate of DCMB was significantly higher than that of brush cytology (81.0 % [34/42] vs. 38.1 % [16/42]; P < 0.001). No severe complications occurred; three patients (7.1 %) experienced mild procedure-related acute pancreatitis. CONCLUSIONS: The novel DCMB technique was a practical, safe, efficient, and low-costing method for diagnosing malignant biliary stricture with a high accuracy rate.

Endoscopic ultrasonography-guided drainage combined with trans-duodenoscope cyclic irrigation technique for walled-off pancreatic necrosis.

BACKGROUND: Endoscopic ultrasonography-guided drainage has been established as a good treatment modality in the management of walled-off pancreatic necrosis, but the unmanageable infection of postoperation is still a thorny problem due to the poor drainage ability for solid necrotic debris only through transmural stent and nasocystic catheter. AIMS: Introduce a novel therapeutic method, namely endoscopic ultrasonography-guided drainage combined with cyclic irrigation technique in managing patients with walled-off pancreatic necrosis. METHODS: 18 patients with severe acute pancreatitis complicated with walled-off pancreatic necrosis received treatment with endoscopic ultrasonography-guided drainage combined with cyclic irrigation were involved in this retrospective study. RESULTS: 17 of 18 patients with walled-off pancreatic necrosis were treated by this new therapeutic method. Subsequent surgery was performed in 1 case due to uncontrolled infection, complications such as perforation, bleeding or multiple organ failure were not observed. Treatment success rate was high (16 in 17, 94.12%). CONCLUSION: Endoscopic ultrasonography-guided drainage combined with cyclic irrigation is an effective treatment option for symptomatic walled-off pancreatic necrosis to facilitate drainage and obviate the need for subsequent surgery or endoscopic necrosectomy.

[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

[Expression of mouse lipopolysaccharide response protein LRP in E. coli and preparation of rabbit anti-mLRP serum].

AIM: To express mouse lipopolysaccharide response protein (mLRP) and prepare rabbit anti-mLRP serum. METHODS: The predicted mouse lrp cDNA sequence was obtained by splicing homologous ESTs by comparing human lrp cDNA with mouse ESTs. Then the primers were designed. mlrp cDNA from NIH3T3 cells stimulated with lipopolysaccharide (LPS) was amplified by RT-PCR and was cloned into prokaryotic expression vector pTAT to construct recombinant expression vector pTAT-mlrp. The His-TAT-mLRP fusion protein was expressed in E. coli BL21(DE3) and was used to immunize the rabbits to get rabbit anti-mLRP serum. The anti-serum was purified by the acetone precipitation method. The specificity of the rabbit anti-mLRP serum was determined by Western blot. RESULTS: The predicted length of mlrp cDNA was 1905 bp. The encoding region of the cloned mlrp cDNA, 1554 bp, was inserted into pTAT. The His-TAT-mLRP fusion protein was expressed successfully in E. coli. The rabbit anti-mLRP serum was prepared by immunizing the rabbit with mLRP protein. CONCLUSION: The successful expression of mLRP and the preparation of rabbit anti-mLRP serum lays the foundation for further study of the function of mLRP.

[Expression of a candidate human Era binding protein A19 and the preparation of its polyclonal antibody].

AIM: To express a candidate hEra binding protein A19 in Escherichia coli and to prepare anti-A19 antibody. METHODS: A19 gene was amplified by PCR from the plasmid containing A19 gene and was cloned into the expression vector pGEX-4T3 which was then transformed into E.coli. The A19 protein was expressed under IPTG induction. Antiserum was prepared by immunizing rabbits with the expressed A19 protein. The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: The expressed A19 accounted for about 30.2% of total bacterial protein. The titer of the antiserum was about 1:4 000. Western blot analysis indicated that the antiserum had high specificity. CONCLUSION: A19 fusion protein was highly expressed. The specific anti-A19 antiserum was prepared successfully.

[Expression and purification of two kinds of alternative splicing mouse Era].

AIM: To express and purify two alternative splicing mouse Era proteins and detect whether anti-human Era antibody can be used in the study of mouse Era proteins. METHODS: Two fusion protein expression vectors, pMAL-meraW and pMAL-meraS, were constructed, then the MBP-mEra proteins were expressed in E. coli. The target proteins were purified by amylose affinity chromatography. The specificity of rabbit anti-human Era antibody to the proteins was identified by Western blot. RESULTS: The expressed MBP-mEraW and MBP-mEraS proteins constituted approximately 17% and 19% of the total bacterial proteins. The purity of the fused proteins was 67% and 61% respectively after amylose affinity chromatography. Rabbit anti-human Era antibody had high specificity to these two kinds of splicing mouse Era proteins. CONCLUSION: Two fusion mera genes could be expressed in E. coli by using gene recombination technique. The high specificity of rabbit anti-human Era antibody to the two splicing mouse ERA proteins indicates that this antibody can be used to study the function of these two kinds of splicing mouse Era.

[Preparation of anti-Red antisera and its subcellular localization].

AIM: To prepare rabbit anti-Red antisera. METHODS: The bet, exo and gam genes of lambda phage were amplified by PCR from genomic DNA and cloned into the expression vector pDH2, respectively. Red proteins were induced to express at 42 degrees C. The expressed proteins were analyzed by PAGE and thin-layer scanning. The antisera were prepared by immunizing rabbits with the three Red proteins, respectively. The titers and specificities of the antisera were detected by Western blot. RESULTS: Beta, Exo and Gam proteins accounted for about 40.3%, 49.2% and 73.4% of total bacterial protein, respectively. The titers of the antisera were about 1:2,000. Western blot analysis indicated that the three antisera all had good specificities to the corresponding proteins. CONCLUSION: Specific anti-Red antisera are prepared successfully.

[Antitumor immune responses induced by gene transfer of 4-1BBL into hepatocellular carcinoma Hepa1-6 in vitro].

OBJECTIVE: To study the cytotoxic activity against tumor cells and cytokines production of spleen cells induced in vitro by murine 4-1BBL gene transfected Hepa1-6. METHODS: The eukaryotic expression vector pCDNA3.1(+)-m4-1BBL was transfected into murine hepatocellular carcinoma cell line Hepa1-6 by Liposomes. Then the transfected cells were selected in medium containing G418 (400 - 800 micro g/ml) and termed as Hepa1-6-m4-1BBL. The TCV-m4-1BBL was obtained by treating them with mitomycin (MMC). Cocultivation TCV with syngeneic murine spleen cells, then the lymphocytes were tested for cytotoxic activity against Hepa1-6-wt cells and the supernatants were harvested for detecting the cytokines (IL-2, TNF-alpha and GM-CSF). RESULTS: Hepa1-6-m4-1BBL cells expressed 4-1BBL protein with highest cell surface level. The 4-1BBL mRNA could still be detected in the cells when cultured 48 h after treated with MMC (80 mg/L). Comparing with TCV-Hepa1-6, the tumor cell vaccine derived from Hepa1-6-m4-1BBL (TCV-m4-1BBL) could induce a more efficient cytotoxic activity of syngeneic murine lymphocyte against its parental tumor cell Hepa1-6 (P < 0.05), but not against non-parental tumor cell H22 and NIH3T3. Higher levels of IL-2, TNF-alpha and GM-CSF were released by the splencytes after stimulated by TCV-m4-1BBL. CONCLUSIONS: These results suggest the expression of m4-1BBL by tumor cells is effective in inducing antitumor immune responses.

[The expression of truncated YggG protein and preparation of its polyclonal antibody].

AIM: To express truncated YggG protein (TYP) in Escherichia coli and to prepare anti-TYPE antibody. METHODS: Truncated yggg gene was amplified by PCR from the plasmid containing full length yggg gene and was cloned into the expression vector pDH2. The expression of TYP was achieved by thermal induction. Antiserum was prepared by immunizing rabbit with TYP,The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: Thin-layer scan analysis showed that TYP accounted for about 37.1% of total bacterial protein. The antiserum was about 1:2,000 in titier and highly specific. Full-length rggG protein could also be recognized by the antiserum. CONCLUSION: The TYP was highly expressed, and specific anti-TYP antiserum was prepared successfully.

[Cloning, expression and antitumor effect of mouse costimulatory molecule 4-1BBL].

AIM: To clone mouse 4-1BBL gene, construct its eukaryotic expression vector, and evaluate antitumor activity of the expression product. METHODS: RT-PCR was used to amplify mouse 4-1BBL gene from total RNA of C57BL/6 splenocytes stimulated by PHA. Then m4-1BBL cDNA was subcloned into eukaryotic expression vector pcDNA3.1(+) and transfected into mouse hepatocellular carcinoma cell line Hepa1-6. The expression of m4-1BBL in transfected cells was detected by RT-PCR, indirect immunofluorescence staining, and flow cytometry. Non-adherent splenocytes from non-immunized C57BL/6 mice were incubated with mitomycin-treated non-transfected Hepa1-6(Hepa1-6-wt) or transfected Hepa1-6 cells (Hepal-6-m4-1BBL), respectively. Then the lymphocytes were tested for cytotoxic activity to Hepa1-6-wt cells. RESULTS: The Hepa1-6 cells transfected by pcDNA3.1(+)-m4-1BBL could efficiently express m4-1BBL. As compared with Hepa1-6-wt cells,Hepa1-6-m4-1BBL cells could induce more efficiently cytotoxic activity of lymphocytes to Hepa1-6-wt cells (P<0.01). CONCLUSION: The expression of m4-1BBL by tumor cells is effective in inducing antitumor immune response.

[Expression of human osteoprotegerin gene in E. Coli and bioactivity analysis of expression product].

OBJECTIVE: To express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro. METHODS: Synthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products. RESULTS: The sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L. CONCLUSIONS: Human OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.

[Construction and biological activity study of human osteoprotegerin expressing adenoviral system].

Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices.

[The OPG/RANKL/RANK system and bone resorptive disease].

The OPG/RANKL/RANK system plays an important role in osteoclastogenesis and represents a great progress in bone biology. RANKL, which expresses on the surface of osteoblast/stromal cells and activated T cells, binds to RANK on the osteoclastic precursors or mature osteoclasts, and promotes osteoclastogenesis and bone resorption. While osteoprotegerin (OPG), which is expressed by osteoblasts/stromal cells, strongly inhibits bone resorption by binding to its ligand RANKL and thereby blocks the interaction between BANKL and RANK. A number of cytokines and hormones exert their effects on bone metabolism by regulating the OPG/RANKL ratio in the bone marrow microenvironment. RANK is also expressed on mammary epithelial cells and RANKL expression in these cells is induced by pregnancy hormones, RANKL and RANK are essential for the formation of the lactating mammary gland and the transmission of maternal calcium to neonates in mammalian species. Modulation of these systems provides a unique opportunity to develop novel therapeutics to inhibit bone loss in osteoporosis, rheumatoid arthritis, and bone metastasis of cancer. Further research should be focused on the cooperation of OPG/RANKL/RANK system with other signal pathways and the interactions among bone remodeling, immune system and endocrinology system. Currently, the development of OPG analogues or compounds which may stimulate OPG expression is becoming an attractive industry which may be profitable to both patients and manufacturers.

[Gene cloning and prokaryotic expression of the PID domain of human DOC-2 molecule].

AIM: To clone the PID domain of human DOC-2 (nDOC-2) and express it in E.coli DH5alpha. METHODS: The cDNA fragment encoding the PID domain of nDOC-2 was amplified by RT-PCR from normal human ovarian tissue and cloned to pUC19. The DNA fragment from the pUC19-nDOC-2 digested with BamH I and EcoR I was ligated to the BamH I/EcoR I digested prokaryotic expression vector pGEX-4T-1.The expression of fusion protein was induced with IPTG and the expressed product was identified by SDS-PAGE. RESULTS: (1)The sequencing and endonucleases digestion analysis showed that the fragment of nDOC-2 gene was insert into vectors pUC19 and pGEX-4T-1;(2)SDS-PAGE showed the nDOC-2 gene had been expressed in E.coli DH5alpha. CONCLUSION: The PID domain of nDOC-2 was expressed successfully in prokaryote, which makes preparation for further researching the function of DOC-2 and preparing antibodies to DOC-2 protein.

[Strategies of functional analysis of new genes].

Functional analysis of new genes is playing a central role in postgenomic era. Here we reviewed several main strategies including bioinformatics, gene transduction, antisense technology, certain gene silence induced by RNA interference (RNAi), transgene and gene knockout and artificial chromosome transduction.

[High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding].

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.

Cloning and Optimized Expression of Human Angiogenin in E.coli.

Angiogenin(ANG) is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors. To study the biological funcption and find the antagonistic drugs of angiogenin, the angiogenin was allowed to be expressed by E.coli. By the aid of computer, the sequence around the start codon of angiogenin gene was modified according to local secondary structure. The modified human ang gene was amplified by reverse-transcription polymerase chain reaction from the human lung cancer cell line A549, and inserted into the prokaryotic expression vector pLDH99. After screening, high expression recombinants were obtained, and the expression level of the hANG was about 30% of total bacteria protein by SDS-PAGE. Biological assays indicated that the rhANG could induce new blood vessel formation in CAM in vitro. Our data showed that the recombinant hANG was active and the optimized expression of ang gene was practicable.