Objective:To express and purify a new fusion protein harboring human epidermal growth factor receptor(EGFR) binding domain and Interleukin-18(IL-18), as well as preliminary assay biological activity of recombinant proteins.Methods:Fusion protein was expressed in insect cells Spodoptera frugiperda cell (Sf9) by using Bac-to-Bac system, and an abbreviation purification procedure was used to purify fusion protein. IFN-?induction assay and EGF Receptor competitive test was used to determine fusion protein's biological activity.Results:SDS-PAGE and western blot assay showed that purified EGF-IL-18 fusion protein had high purity in 20 kD as expected and had the same antigenic specificity as human IL-18. IFN-?induction assay and EGF Receptor competitive test showed that fusion protein induced production of IFN-?in human PBMC and bound with tumor cells.Conclusion:EGF-IL-18 fusion protein has been successfully expressed and purified in insect cells and shows potential to apply in targeting therapy for tumor.

Objective:To develop a real-time PCR assays based on TaqMan chemistry for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.Methods:The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold series diluted primary viral stocks were used for plaque assay and DNA extraction.Bacmid(baculovirus plasmid) was 10-fold series diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.Results:The standard linear(101 to 108 copies) from quantitation was achieved with the standard curve.We also find that the "vg/ml" titer value is generally about 10 times than "pfu/ml" titer of the same recombinant virus stock.Conclusion:A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the "vg/ml" titer of virus.The method is rapid and quantitative over a wide range of virus titers.

AIM: To investigate the alteration of serum free fatty acids in the carbon tetrachloride-induced fatty liver. METHODS: Drug-induced fatty liver rat models were built by injection 40% CCl_4. Serum free fatty acids were analyzed by gas chromatography. RESULTS: In the composition of serun free fatty acids, polyunsaturated fatty acids [oleic acid C18∶1,(28.672?7.332 ?/mg?L~(-1) vs 41.373?2.180 ?/mg?L~(-1)), linoleic acid C18∶2(16.739?0.871 ?/mg?L~(-1) vs 24.959?5.325 ?/mg?L~(-1)), arachidonic acid C20∶4(6.105?2.656 ?/mg?L~(-1) vs 9.802?0.779 ?/mg?L~(-1)),P

Carotid Artery, Internal ; diagnostic imaging ; Cerebrovascular Disorders ; diagnostic imaging ; physiopathology ; Extremities ; physiopathology ; Humans ; Ischemic Attack, Transient ; complications ; diagnostic imaging ; drug therapy ; Male ; Middle Aged ; Middle Cerebral Artery ; diagnostic imaging ; Thromboembolism ; diagnostic imaging ; physiopathology ; Ultrasonography, Doppler, Transcranial

Objective:To improve the primary culture method of rat pulmonary microvascular endothelial cells(PMVECs) and obtain purified PMVECs.Methods:The modified tissue block pasted culture method was used to isolate and culture Wistar rat PMVECs. The morphous of cultured cells were observed by microscopy. The cultured cells were identified by detecting factor Ⅷ related antigen and binding isolectin B4. Results and Conclusion:The morphous of cultured primary PMVECs in vitro showed short fusiform shape or polygon, and the monolayer of cultured cells displayed the shape of pavingstone. But the morphous changed followed the transfer of culture and the change of culture condition. The cultured cells had characterization of binding isolectin B4 and negative immunocytochemical staining for factor Ⅷ related antigen. The cultured PMVECs have good growth state and purity,and can be subcultringed stably.The observation of cell morphous integrating with immunocytochemical staining is a reasonable identification method for PMVECs.

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT. [

Objective To establish a method of isolation, purification, primary culture and identification of alveolar type Ⅱ epithelial cells(AT-Ⅱ).Methods The AT-Ⅱs were isolated from Wistar rats by trypsin,purified by differential centrifugation, erythrocyte spallation, differential adherence and immune adherence, and identified by observing the morphology of cultured cells under the inverted phase and tannic acid staining. Results and Conclusion The cultured primary AT-Ⅱs in vitro presented single or island form growth, and their shapes were round or elliptical. A great deal of fine particles showed sharp contrast, and were observed in intracytoplasm. The cell nuclei were clear. They were positive for tannic acid staining.The primary culture AT-Ⅱs obtained from improved isolation and purification have good growth state and purity, and are suitable for research in vitro.

Objective: To investigate the creation of smoke-free environments in public places in Hangzhou City, so as to provide insights into effective implementation of the tobacco control policy. Methods: The party and government administrations at each level, medical institutions, educational places, restaurants and entertainment places, and open public places were enrolled. The creation of smoke-free environments was investigated in these places through undercover investigation with field observations and concealed photography by a third-party professional investigation company from November to December, 2022. The building of smoke-free environments (totally 60 scores) and no smoking indoors (totally 40 scores) were evaluated according to the Criteria for Scoring of Smoke-free Organizations in Hangzhou City. Results: Totally 909 places were investigated, and the comprehensive score of smoke-free environment building was (82.83±14.13) points. There were 285 party and government administrations with a comprehensive score of (84.19±12.85) points, 65 medical institutions with a comprehensive score of (90.35±6.95) points, 65 educational places with a comprehensive score of (83.43±16.81) points, 403 dining and entertainment places with a comprehensive score of (80.68±14.75) points, and 91 open public places, with a comprehensive score of (82.34±14.77) points. There were 397 places with standardized tobacco control tips at entrances (43.67%), 308 places with tobacco control signs posted as required (33.88%), 707 places that set outdoor smoking areas correctly (77.78%), 68 places with smoking paraphernalia (7.48%), 28 places with tobacco sales (3.08%). There were 732 places without signs of indoor smoking (80.53%), 850 places without indoor smoking (93.51%) and 24 places without dissuading from smoking (2.64%). Conclusion The indoor no-smoking is overall satisfactory in public places in Hangzhou City; however, standardizing no-smoking tips at entrances, standardizing the posting of no-smoking signs and assignment of tobacco control materials remain to be improved.

Insect larvae and adult insects found on human corpses can provide important forensic evidence however it is useful to be able to prove evidence of association. Without this, it could be claimed that the insect evidence was a contaminant or had been planted on the body. This paper describes how mitochondrial DNA (mtDNA) and STR analysis of the crop contents of larvae of the blowfly Aldrichina grahami collected from separated body parts was used to provide evidence of association.

INTRODUCTIONIntravenous thrombolysis has been shown to improve outcome after acute cerebral infarction if given within 3 hours of symptom onset. There are no data in Singapore on the timing of hospital presentation after acute cerebral infarction as well as factors and reasons for delayed presentation.

MATERIALS AND METHODSAs intravenous thrombolysis has recently been licensed for use in acute cerebral infarction in Singapore, we studied 100 consecutive acute cerebral infarction admitted to the Singapore General Hospital for timing of hospital presentation, reasons associated with delay in presentation and hypothetical acceptance of intravenous thrombolysis.

RESULTSOnly 9% of patients presented to hospital within 2 hours of symptom onset. Factors associated with hospital presentation within 2 hours were a large stroke and lack of pre-hospital consultation. Failure to recognise the severity of symptoms and inability to seek medical attention unaided were the 2 most common reasons for delayed presentation. One-third of patients or their relatives hypothetically would accept intravenous thrombolysis, suggesting that a thrombolysis service is feasible at the Singapore General Hospital. However, it would be hindered by the low proportion of patients who present early to hospital after symptom onset.

CONCLUSIONOur results support the need for a public education programme to highlight the identification of stroke symptoms and the need to present to hospital as soon as possible after the onset of stroke symptoms.

Acute Disease ; Aged ; Cerebral Infarction ; drug therapy ; physiopathology ; Emergency Service, Hospital ; Female ; Fibrinolytic Agents ; therapeutic use ; Hospitals, General ; Humans ; Infusions, Intravenous ; Male ; Middle Aged ; Patient Acceptance of Health Care ; statistics & numerical data ; Prospective Studies ; Singapore ; Time Factors ; Treatment Outcome