Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.

OBJECTIVETo explore the underlying mechanism of mesenchymal stem cells (MSCs) transfer induced cardiac function improvement in failing hearts.

METHODSCongestive heart failure (CHF) was induced in rats by cauterization of the heart wall. MSCs were cultured from autologous bone marrow and injected into the border zone and the remote myocardium 5 days after cauterization.

RESULTSTen weeks later, cardiomyocyte nucleus mitotic index, capillary density and expression of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were significantly increased in the border zone and significantly reduced in the remote myocardium in CHF rats (all P<0.05 vs. sham). Besides cardiac function improvement and left ventricular remodeling attenuation evidenced by hemodynamic and echocardiographic examinations, expressions of IGF-1, HGF and VEGF in the remote myocardium and in the border zone were also significantly upregulated (P<0.05 or P<0.01 vs. CHF), and cardiomyocyte nucleus mitotic index as well as capillary density were significantly increased in CHF rats with MSCs (P<0.05 or P<0.01 vs. CHF). Moreover, collagen area was significantly reduced and myocardial area was significantly increased in the border zone in these rats too.

CONCLUSIONAutologous MSC implantation upregulated expressions of growth factors enhanced cardioangiogenesis which might be the underlying mechanisms for improved cardiac function and attenuated left ventricular remodeling induced by MSCs transplantation in failing rat myocardium.

Animals ; Disease Models, Animal ; Heart Failure ; metabolism ; therapy ; Hepatocyte Growth Factor ; metabolism ; Insulin-Like Growth Factor I ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transplantation, Autologous ; Vascular Endothelial Growth Factor A ; metabolism ; Ventricular Remodeling

OBJECTIVETo investigate the protective mechanism of geniposide, baicalin and berberine on hypoxia and reoxygenation injury in cultured rat cerebral microvascular endothelial cells.

METHODA model of four hours hypoxia and twelve hours reoxygenation injury in rat cerebral microvascular endothelial cells in vitro was established. The injured cells were treated with geniposide (0.128, 0.064, 0.032 mmol x L(-1)), baicalin (0.028, 0.014, 0.007 mmol L(-1)) and berberine (0.024, 0.012, 0.006 mmol L(-1)), respectively. The immunocytochemical method and techniques of image quantitative analysis were used to detect the mean optical density and mean area in order to match the protein expression of VCAM-1. The method of RT-PCR was adopted to observe and match the mRNA expression of VCAM-1.

RESULTAs compared with the normal group, the mean optical density, the mean area and the mRNA expression of VCAM-1 of model group were significant increased (P < 0.01, P < 0.01, P < 0.01). As compared with the model group, both the mean optical density and the mean area of all treated groups were decreased, and there was significant difference between them (P < 0.01, P < 0.01). As compared with normal group, the mean optical density of baicalin (0.007 mmol x L(-1)) and berberine (0.012, 0.006 mmol x L(-1)) were significant decreased (P < 0.05, P < 0.01, P < 0.01), but there was no significant difference between the other groups and the normal group. As compared with normal group, the mean area of baicalin (0.0014 mmol x L(-1)) was significant decreased (P < 0.05), but there was significant difference between the other groups and the normal group. The mRNA expression of all treated groups was not only lower than that of the model group but also higher than that of the normal group (P < 0.05, P < 0.05).

CONCLUSIONThe results suggest that geniposide, baicalin and berberine, which are effective compositions of huanglian jiedu decoting, can protect hypoxia-reoxygenation injuried rat cerebral microvascular endothelial cells. One of the protected mechanisms is that they can inhibit the expression of VCAM-1.

Animals ; Berberine ; pharmacology ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Cerebrum ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Flavonoids ; pharmacology ; Gene Expression ; drug effects ; Humans ; Hypoxia ; drug therapy ; genetics ; metabolism ; Iridoids ; pharmacology ; Male ; Oxygen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; genetics ; metabolism ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVETo investigate the protective effect of geniposide, baicalin and berberine for the rat cerebral microvascular endothelial cell.

METHODThe model of hypoxia and reoxygenation injury in rat cerebral microvascular endothelial cells in vitro was established. Both normal and model cells were treated with geniposide (1.024, 0.512, 0.256, 0.128, 0.064, 0.032, 0.016, 0.008 micromol x mL(-1)), baicalin (0.224, 0.112, 0.056, 0.028, 0.014, 0.007, 0.003 micromol x mL(-1)) and berberine (0.192, 0.096, 0.048, 0.024, 0.012, 0.006, 0.003 micromol x mL(-1)). Cell activity was measured by methyl thiazolyl tetrazolium (MTT) test.

RESULTAfter hypoxia/hypoglycemia cultures for 4 hour and reoxygenation for 12 hour, geniposide (0.128, 0.064, 0.032 micromol x mL(-1)), baicalin (0.028, 0.014, 0.007 micromol x mL(-1)) and berberine (0.024, 0.012, 0.006 micromol x microL(-1) could protect the injuried cerebral microvascular endothelial cells.

CONCLUSIONAppropriate concentration of geniposide, baicalin and berberine, which are effective components of Huanglian Jiedu decoction, could protect the injuried cerebral microvascular endothelial cells.

Animals ; Berberine ; isolation & purification ; pharmacology ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; blood supply ; Dose-Response Relationship, Drug ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; Endothelial Cells ; cytology ; drug effects ; Flavonoids ; isolation & purification ; pharmacology ; Iridoids ; isolation & purification ; pharmacology ; Male ; Neuroprotective Agents ; pharmacology ; Oxygen ; pharmacology ; Plants, Medicinal ; chemistry ; Pyrans ; isolation & purification ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the protective mechanism of geniposide, baicalin and berberine on hypoxia and reoxygenation injury in cultured rat cerebral microvascular endothelial cells.

METHODTo establish a model of hypoxia four hours and reoxygenation twelve hours injury in rat cerebral microvascular endothelial cells in vitro. The injured cells were treated with geniposide (0. 128, 0.064, 0.032 micromol mL(-1), baicalin (0.028, 0.014, 0.007 micromol mL(- 1)) and berberine (0.024, 0.012, 0.006 micromol mL(-1)). The expression of p65 subunit of NF-kappaB was detected by immunocytochemical assay and techniques of image quantitative analysis. The protein expression of NF-kappaB was calculated with the mean optical density and mean area. The nuclear translocation of NF-kappaB was calculated with the percentage of positive cells and ratios of light transmittance of cytoplasm and cell nucleus.

RESULTCompared with the normal group, both the protein expression and the nuclear translocation of NF-kappaB of model group were significant increased (P <0.01). Compared with the model group, the mean optical density of all treated groups was decreased ,but these was no significant difference between them. As compared with model group, the mean area of all treated groups was significant decreased (P < 0.01). The percentage of nuclear translocation of all treated groups is not only lower than that of the model group but higher than that of the normal group (P <0.01). Compared with the model group, the ratios of light transmittance of cytoplasm and cell nucleus of all treated groups was significantly elevated (P <0.01).

CONCLUSIONThe results suggesed that geniposide, baicalin and berberine could protect hypoxia/reoxygenation injuried rat cerebral microvascular endothelial cells injury. One of the mechanism may lie in inhibiting both the protein expression and the nuclear translocation of NF-kappaB.

Animals ; Brain ; blood supply ; Cell Nucleus ; drug effects ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Hypoxia ; complications ; Male ; Microvessels ; pathology ; NF-kappa B ; metabolism ; Oxygen ; metabolism ; Protein Transport ; drug effects ; Rats

BACKGROUNDAlthough severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA.

METHODSSARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens.

RESULTSThe sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001).

CONCLUSIONN199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.

Amino Acid Sequence ; Animals ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Molecular Sequence Data ; Nucleocapsid Proteins ; blood ; genetics ; Sensitivity and Specificity ; Severe Acute Respiratory Syndrome ; diagnosis ; Swine

As a natural product with a long history of medicinal use, parthenolide has aroused great interest of chemists and biologists. Existing studies have shown that it has anti-inflammatory, antitumor and other pharmacological activities, and also revealed its action on NF-κB signaling pathway, DNMT1 enzyme and Wnt/β-catenin signaling pathway. But its biological targets remain to be elucidated systematically. Proteolysis Targeting Chimeras (PROTAC) provides a new strategy for target discovery of natural products, which can be used to explore the panorama of protein changes in cells through proteomic investigation, so as to analyze their potential targets. Based on this idea, current study designed and synthesized 20 parthenolide-derived degraders. After measured their antitumor activity in vitro, selected compounds were carried out the proteomic experiment. Finally, 139 down-regulated differentially expressed proteins were identified and the discovery of parthenolide interacting protein was preliminarily explored.

IMM0306 is a recombinant human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein, intended to treat refractory or recurrent CD20 positive B-cell non-Hodgkin lymphoma (B-NHL) in clinical. In this study, an ELISA method was established to evaluate the pharmacokinetics of IMM0306 in human serum. The experiment was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (No. CTR20192612). Recombinant human CD47 protein was coated with the plate overnight, blocking the plate with 5% skin milk for 2 h. After washing, 100 μL per well standard and unknown samples were added, and incubated for 1.5 h. After washing, the detection antibody Anti-IMM0306-Biotin was added and incubated for 1 h, and then HRP-labeled streptavidin was added for 1 h, and the color was detected. The optimal concentration of coating reagent was 2 μg·mL^-1 by ELISA method, and the optimal dilution of anti-IMM0306-biotin and SA-HRP were 1∶500 and 1∶5 000, respectively. The lower limit of quantitation was 4 ng·mL^-1, and the standard curve range was 4-100 ng·mL^-1. The verification results of the method meets the corresponding acceptance criteria, and can be used in IMM0306 clinical pharmacokinetic studies.

OBJECTIVEIn this study, we aimed at exploring the association between work-related musculoskeletal disorders (WMSDs) and work organization based on a case-control study.

METHODSA total of 1938 workers who claimed to suffer from WMSDs were selected from Beijing, Henan, Hubei, and the Guangdong province. The control group consisted of 2009 workers employed in similar industries without severe disease or musculoskeletal discomforts. We used a modified version of the questionnaire developed by the NMQ and the DMQ to investigate individual and work-related factors.

RESULTSA total of 13 variables (P<0.1) were selected by the chi-square test and finally, 7 variables entered into the equation, with 6 variables reaching statistical significance (P<0.05). The odds ratios (OR) of 'work changing with season' and 'sufficient rest time' did not reach 1 (0.749 and 0.441, respectively). In addition, 'sufficient rest time' seemed to be the stronger protective factor according to its higher standardized coefficient. And 'repetitive work every minute', 'constantly repetitive work' (every day), 'shortage of site personnel', and 'often switching shifts with others' seemed to be the risk factors.

CONCLUSIONWork organization may have comprehensive effects on the occurrence of WMSDs. This pattern of associations suggests that further investigation into the mechanism of how work organization affects the prevalence of WMSDs is required.

Adult ; Case-Control Studies ; Chi-Square Distribution ; China ; epidemiology ; Cross-Sectional Studies ; Cumulative Trauma Disorders ; epidemiology ; etiology ; Demography ; Ergonomics ; standards ; Female ; Humans ; Logistic Models ; Male ; Musculoskeletal Diseases ; epidemiology ; etiology ; Occupational Diseases ; epidemiology ; etiology ; Personnel Staffing and Scheduling ; organization & administration ; Prevalence ; Risk Factors ; Seasons ; Surveys and Questionnaires ; Work Schedule Tolerance ; physiology ; Workplace ; organization & administration ; Young Adult

BACKGROUNDAcupuncture can not only be used for well-known diseases, but also for so-called modern lifestyle-related diseases. Using innovative methods like e.g. new analyses of heart rate variability (HRV), it is also possible to investigate diseases like burnout syndrome, ie., qi deficiency in Chinese medicine (CM).

OBJECTIVEThe main object of this research protocol is to perform research on the relationship of burnout syndrome and heart rate (HR) and HRV.

METHODSA total of 175 patients with burnout syndrome (qi deficiency syndrome) in five groups and 35 healthy volunteers will be investigated. Based on random numbers generated by computer and concealed in opaque envelops, the patients will be assigned to four acupuncture groups using Zusanli (ST 36) acupuncture stimulation, Guanyuan (CV4) acupuncture stimulation, both points, and both points with Streitberger device respectively, and a moxibustion group using both points mentioned above, with 35 cases in each group. Altogether four different experiments are planned. Experiment 1 includes 70 subjects and is a comparison between a burnout group and a control group (healthy volunteers). The evaluation parameters are different scores and indices of HR and HRV. Experiment 2 includes 140 subjects and compares the efficacy of different acupuncture points. In experiment 3 (105 subjects), acupuncture and moxibustion should be compared to healthy volunteers. Experiment 4 (70 subjects) investigates the long-term therapeutic effects of acupuncture and moxibustion on the scores of qi deficiency and HR/HRV in qi deficiency patients. In both the acupuncture and moxibustion groups, a total of 10 treatments will be performed.

CONCLUSIONSThe joint research aims at the scientific evaluation of CM, mainly in the field of HRV. This parameter could be a very good indicator of the state of health and can be inflfluenced by different acupuncture methods, as shown in the past.

Acupuncture Therapy ; Adult ; Austria ; Burnout, Professional ; therapy ; China ; Female ; Heart Rate ; physiology ; Humans ; Life Style ; Moxibustion ; Research Design