OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Animals ; Antibodies ; Capsid ; Capsid Proteins/genetics* ; Dependovirus/genetics* ; Prokaryotic Cells ; Rabbits ; Recombinant Proteins/genetics*

Studies have found that metformin is not only the preferred drug for lowering blood sugar, but also shows lipid-lowering and weight-loss effects. The purpose of this study was to use a hyperlipidemia hamster model to investigate the lipid-lowering effect of metformin and its effect on important metabolic pathways in lipid metabolism disorders. Fifty golden hamsters were divided into a control group, a model group, metformin high- and low-dose groups, and a simvastatin group. A high-fat diet was fed for 1 week to create the model, and then drug was administered for 11 weeks with the high-fat diet. Serum was taken for measurement of blood lipid and blood glucose at 2, 6, and 9 weeks after administration, and at weeks 3, 5, and 9 feces and urine were collected for ¹H NMR metabolomics tests. After 11 weeks of intravenous injection of [U-¹³C₆] glucose, serum was collected for a ¹³C NMR metabolic flux test. The results showed that the administration of metformin can significantly reduce blood lipids and glucose levels and can significantly affect metabolic pathways such as sugar metabolism, lipid metabolism, ketone metabolism, amino acid metabolism, and intestinal flora metabolism. The results of the metabolic flux analysis showed that the high-fat diet reduced the metabolism of tricarboxylic acids by 37.48%. After administration of low and high doses of metformin the metabolism of tricarboxylic acid increased by 98.14% and 143.10%, respectively. After administration of simvastatin tricarboxylic acid metabolism increased by 33.18%. The results indicate that metformin has a significant effect on promoting energy metabolism. This study used a combination of metabolomics and metabolic flow to explore the effect of metformin on lipid metabolism disorders and quantifies changes in the key pathway of energy metabolism-the tricarboxylic acid cycle. This study provides useful information for the study of the efficacy and mechanism of metformin, as well as a practical technical method for the screening of lipid-lowering drugs based on a hamster model.

The chemical constituents of Rehmanniae Radix Preparatawere prepared according to the traditional method of "jiu zheng jiu shai" and investigated using multiple chromatographic methods. Six alkaloids were isolated and their structures were elucidated from spectral data and physicochemical properties, as follows: rehmanniae alkaloid A (4-{[(5-O-á-D-galactopyranosyloxy)methyl]-1H-pyrrole-2-carbaldehyde-1-yl}butyric acidmethyl ester) (1), baimantuoluoamide B (2), capparisine C (3), harman-3-carboxylic acid (4), (2S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-carboxylate (5), and 1-[2-(furan-2-yl)-2-oxoethyl]pyrrolidin-2-one (6). Among them, compound 1 is a new alkaloid. Compounds 2-6 were newly isolated from Rehmannia glutinosa Libosch.The effect of compounds 1-6 on NRK-52e cell injury induced by LPS was investigated. The results show that compounds 1-3 exhibit protective effects against LPS-induced damage to NRK-52e cells.

A highly sensitive and selective bioluminescent probe for hydrazine (BPH) was designed, synthesized and evaluated for detection of hydrazine in vitro and in vivo. BPH was designed to include a specific recognition group (acetyl) of hydrazine at an appropriate modification site of the optical reporter hydroxyluciferin (D-luciferin), which showed excellent performance both in selectivity and sensitivity to hydrazine. The results showed that the bioluminescent probe BPH developed in this study is an innovative and widely applicable tool for detecting hydrazine in complex natural environment or in animals.

Objective:To analyze whether the OAZI-1 (ornithine decarboxylase antizyme inhibitor-1) protein complex isolated from tumor cells could induce specific antitumor effects in the experiment mice .Methods:OAZI-1 protein complexes were isolated from B16-F1 melanoma cells by immune magnetic beads coated with OAZI-1 antibody and used as the vaccine to immune the C 57BL/6 mice.After immunization,the mice were inoculated subcutaneously with live B 16-F1 cells and then tumor formation and growth were ob-served.ELISA was used to determine the level of cytokine IFN-γin the serum of immunized mice.Lactate dehydrogenase assay (LDH) was performed to evaluate killing effect of spleen lymphocytes on B 16-F1 cells.The mice immunized by purified OAZI-1 from prokaryotic expression and PBS were used as controls in the animal experiment .Results: Compared with the control mice ,the spleen lymphocytes ( effector cells ) from the mice inoculated with OAZI-1 protein complexes had stronger killing ability on B 16-F1 cells (target cells).At three different effector:target ratio (10:1,50:1,100:1),the killing ability of these spleen lymphocytes were 46.2%, 59.5%and 92.5% respectively,which was significantly higher than the spleen lymphocytes from the mice inoculated with purified AZIN-1 protein (36.1%,26.8% and 45.9%) or inoculated with PBS (24.6%,24.0% and 27.2%).In addition,the content of serum anti-tumor cytokine IFN-γwas also significantly higher in the mice inoculated with OAZI-1 protein complexes (538.3 pg/ml) than the mice inoculated with purified AZIN-1 ( 256.2 pg/ml ) or with PBS ( 131.0 pg/ml ) .When B16-F1 live cells were subcutaneously inoculated into the immunized mice described above ,the tumor formation rate was only 40%in the mice immunized with OAZI-1 protein complex ,but 100%in the mice immunized with PBS or purified OAZI-1.The growth of inoculated tumors in the mice immunized with OAZI-1 protein complex was also much slower than the control mice .Conclusion:The results in this study suggest that the OAZI-1 protein complex isolated from B 16-F1 tumor cells could contain some tumor antigens .When used as tumor vaccine to inoculate mice ,this complex can induce anti-tumor immune killing activity in experimental animals .

BACKGROUNDStatins and ezetimibe have been reported to change the balance of cholesterol metabolism, but few studies have been performed on Chinese patients. The aim of this study was to evaluate changes in cholesterol metabolism markers in patients with coronary heart disease.

METHODSForty-five patients with coronary heart disease were treated with 20 mg/d of simvastatin for four weeks. Subjects were then divided into two different therapy groups according to whether they reached the target values for total cholesterol and low density lipoprotein cholesterol level. Patients who reached the target values remained on simvastatin and those who did not reach the target values took a combination of simvastatin plus 10 mg/d ezetimibe until the 12th week. The concentrations of cholesterol synthesis markers (lathosterol and desmosterol) and absorption markers (campesterol and sitosterol) were measured on the 1st, 4th, and 12th week of the study by gas chromatography.

RESULTSAfter treatment with simvastatin for four weeks, the levels of total cholesterol and low density lipoprotein cholesterol decreased significantly compared to levels measured during the 1st week (P < 0.05). On the 12th week the levels of total cholesterol and low density lipoprotein cholesterol had decreased significantly (P < 0.001) compared to levels during the 4th week. By the 12th week the levels of campesterol and sitosterol in the combination group had decreased significantly (P < 0.05) compared with levels measured during the 4th week.

CONCLUSIONSCoronary heart disease patients with high cholesterol synthesis at baseline might gain a greater benefit from simvastatin treatment. Combination therapy with simvastatin plus ezetimibe in patients with low cholesterol synthesis at baseline might increase the success rate of lipid-lowering through decreasing the absorption of cholesterol.

Adult ; Aged ; Azetidines ; administration & dosage ; Cholesterol ; metabolism ; Cholesterol, LDL ; blood ; Coronary Disease ; drug therapy ; metabolism ; Drug Therapy, Combination ; Ezetimibe ; Female ; Humans ; Male ; Middle Aged ; Simvastatin ; administration & dosage

BACKGROUNDAvailable drug-eluting stents (DES) have achieved great success in reducing restenosis rates. Recently, investigators have demonstrated that the durable polymer carrier plays a significant role in DES-related hypersensitive reaction and delays vessel healing. TIVOLI stent is a novel sirolimus-eluting coronary stent with biodegradable coating containing sirolimus and polylactic-co-glycolic acid (PLGA) polymer. The present study sought to evaluate the effectiveness and safety of the TIVOLI biodegradable-polymer-based sirolimus-eluting stent in treating patients with coronary artery disease.

METHODSA prospective, multicenter clinical trial comparing TIVOLI biodegradable coated sirolimus-eluting stent with ENDEAVOR zotarolimus-eluting stent was conducted in 324 patients (TIVOLI group: 168 patients; ENDEAVOR group: 156 patients) at 12 centers in China to demonstrate the non-inferiority of in-stent late loss with TIVOLI stent compared to ENDEAVOR stent in subjects with a maximum of two de novo native coronary artery lesions (lesion length ≤ 40 mm, reference vessel diameter 2.25-4.00 mm). The primary end point was angiographic in-stent late loss at 8-month. The secondary end points were clinical outcomes at 2 years, including major adverse cardiac events (cardiac death, myocardial infarction, or target-lesion revascularization) and stent thrombosis.

RESULTSAngiographic late lumen loss at 8 months in the TIVOLI group was superior to the ENDEAVOR group (in-stent (0.25 ± 0.33) mm vs. (0.57 ± 0.55) mm, diff (95%CI) -0.23 (-0.32, -0.14), P < 0.0001; in-segment (0.25 ± 0.33) mm vs. (0.42 ± 0.55) mm, diff (95%CI) -0.13 (-0.23, -0.02), P = 0.0083). The rate of in-stent binary restenosis at 8 months was reduced from 8.6% in the ENDEAVOR group to 2.9% in the TIVOLI group (P = 0.0229). Compared to ENDEAVOR stent, TIVOLI stent resulted in a significant reduction in target-lesion revascularization (4.2% vs. 9.6%, P = 0.0495) at 2 years. The two-year major adverse cardiac events (MACE) rate was lower for the TIVOLI group, but not significantly different (6.6% vs. 10.9%, P = 0.1630).

CONCLUSIONSTIVOLI was superior to ENDEAVOR stent with respect to late lumen loss at 8 months, and it yielded both lower rates of angiographic binary restenosis at 8 months and target lesion revascularization (TLR) at 2 years. The MACE rate at 2 years was comparable in both groups.

Aged ; Angioplasty, Balloon, Coronary ; methods ; Coronary Angiography ; Coronary Artery Disease ; drug therapy ; therapy ; Drug-Eluting Stents ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Polymers ; chemistry ; Sirolimus ; analogs & derivatives ; therapeutic use ; Treatment Outcome

OBJECTIVETo observe the expression pattern of bone morphogenetic protein receptor IA (BMPR IA) in rats after contusive spinal cord injury.

METHODSThe expressions of BMPR IA, IB, and II were detected by immunochemistry in the spinal cord of normal adult rats, and the expression of BMPR IA was detected in the infinite horizons impactor model at 1, 3, 7, 14, 30, and 60 days after spinal cord injury.

RESULTSIn the spinal cord of normal adult rats, BMPR IA and II were expressed predominantly in the oligodentrocytes and neurons in the grey matter, and also in some astrocytes and numerous microglia cells. Only a low level of BMPR IB expression was detected in the neurons of the grey matter. After spinal cord injury, the expression of BMP IA markedly increased with sustained strong expression in the astrocytes till one month after the injury; its expression was also increased obviously in the microglia cells activated by the injury.

CONCLUSIONThe expression of BMPR IA increases significantly in the astrocytes and activated microglia cells in rats after contusive spinal cord injury, suggesting the involvement of BMP signaling pathway in the physiological and pathological role of glia cells.

Animals ; Astrocytes ; metabolism ; Bone Morphogenetic Protein Receptors, Type I ; metabolism ; Female ; Microglia ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism

BACKGROUNDUnplanned extubation is associated with adverse outcomes in intensive care unit. The massive burn patient differs from other critically ill patients in many ways. However, little is known about the unplanned decannulation (UD) in Burn Intensive Care Unit. This paper describes the special features of the circumstances and outcome of UD of tracheotomy tube in massive burn patients.

METHODSA case series study was performed between January 1999 and December 2008 and UD of tracheotomy tube was analyzed retrospectively. A total of 21 patients with 29 UD events were identified. Demographic data, diagnosis, intervention, UD events and outcome of UD patients were collected. Differences in proportions were compared using the chi-square (χ(2)) or Fisher's exact test.

RESULTSPatients with UD were often burned with head and neck (67%) and combined with inhalation injury (62%). The majority of them (76%) were transferred patients, occurred early (55%) and were accidental UD (79%). UD events tended to happen in day shift (90%) and to be associated with the medical procedure that was performing by caregivers at besides (79%). Loose of the stabilizing rope, medical procedure and tracheotomy malposition were the main causes of UD. Early UD and reintubation failure were associated with patients' death.

CONCLUSIONSUD happened to massive burn patients can lead to patient death. Careful management of respiratory tract was essential for massive burn patients.

Adult ; Burns ; mortality ; surgery ; Device Removal ; adverse effects ; mortality ; Female ; Humans ; Intensive Care Units ; statistics & numerical data ; Intubation, Intratracheal ; Male ; Middle Aged ; Retrospective Studies ; Tracheotomy ; adverse effects

OBJECTIVETo develop an alternative method for assessment of gene delivery systems in vivo.

METHODSMouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.

RESULTSAs little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.

CONCLUSIONSGluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

Animals ; Arecaceae ; enzymology ; Cell Line ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; Mice