OBJECTIVE:To establish a method for simultaneous determination of 5 effective components in Luohua zizhu dry extract. METHODS:UPLC-MS/MS was conducted. The separation was performed on an Zorbax Eclipse Plus C18 column with mo-bile phase of acetonitrile-water(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was set at 40℃and sam-ple size was 2 μL. The analytes were detected in the multiple reaction monitoring(MRM)mode. Nitrogen was used as drying gas and atomized gas. The temperature and flow rate of drying gas were 325 ℃ and 6 L/min. The pressure of atomized gas was 45 psi. The temperature and flow rate of sheath gas were 350℃and 12 L/min. The voltage of capillary were 4000 V(+)and 3500 V(-). The voltage of nozzle was 500 V. RESULTS:The linear ranges of luteoloside,acteoside,quercetin,luteolin and rutin were 0.5048-252.4 ng/mL(r=0.9999),0.7124-356.2 ng/mL(r=0.9990),0.5094-254.7 ng/mL(r=0.9962),0.3030-151.5 ng/mL(r=0.9998) and 0.6022-301.1 ng/mL(r=0.9996),respectively. RSDs of precision,stability and reproducibility tests were all less than 3.0%. The limit of quantitation were 0.42,0.87,0.33,0.12,0.76 ng/mL. The recoveries were 97.99%-101.20%(RSD=1.3%,n=6), 96.50%-101.20%(RSD=1.7%,n=6), 94.81%-99.34%(RSD=1.7%,n=6), 97.54%-100.51%(RSD=1.2%,n=6), 93.37%-98.70%(RSD=1.9%,n=6),respectively. CONCLUSIONS:The method is simple,precise,stable and reproducible, and can be used for simultaneous determination of 5 effective components in Luohua zizhu dry extract.

Objective:To evaluate the application value of robost optimization of brachytherapy for cervical cancer.Methods:Twenty patients who completed radical treatment were recruited in this study. The dose volume histogram (DVH) parameters were statistically compared between the conventional and robust optimization plans, and the robustness between the conventional and robust optimization plans was evaluated using DVH and DVH bands. The robust optimization method utilized the worst dose distribution to consider the dose in the presence of uncertainties. In each optimization iteration, the dose distributin when the radioactive source shifted along the X, Y, and Z directions (±2 mm), and the dose distribution when the radioactive source was not shifted were calculated. The worst dose distribution for each voxel was the lowest dose in the target and the highest dose outside the target under all circumstances. The iterative objective function was calculated by the worst dose distribution.Results:In the scenario of no shifting of radioactive source position, the mean value of robust optimization was significantly lower and that of V 150% was significantly higher than those of conventional optimization (both P<0.05). When considering the shifting of radioactive source position, the worst dosimetric parameters of multiple dose distributions were statistically compared. The mean HR-CTV D 100% values did not significantly differ between the robust and conventional optimization plans, whereas the mean D 90% value (range: 0.02-0.03 Gy) of robust optimization was significantly higher than that of conventional optimization ( P<0.05). Robust optimization increased the D 2cm 3 of the bladder and small intestine, and the rectum dose was increased with the shifting of the radioactive source position in the robust optimization. The DVH bands did not significantly differ between the conventional and robust optimization plans for all patients. Conclusions:Robust optimization based on the worst dose distribution fails to significantly improve the robustness of brachytherapy for cervical cancer. Alternative methods are required to minimize the dosimetric effect of uncertainties in brachytherapy.

ObjectiveTo verify the reliability of the mouse model of cerebral cortical microinfarct induced by two-photon microscopy and to explore its pathological changes.MethodsSeventeen male C57BL/6J mice were randomly divided into a microinfarct group (n=11) or a sham operation group (n=6).A thinned cranial window of 3 mm diameter was performed over the cerebral cortex with a high-speed micro-drill until the small blood vessels were clearly observed under a dissecting microscope.Then, a permanent single cortical penetrating arteriole occlusion was induced with a gradually enhanced ultrashort laser irradiation through the thinned cranial window with two-photon microscopy.At 7 days after modeling, the cerebral microinfarct volume was measured with HE staining, and the neuron loss, activation of glial cells and deposition of 3-nitrotyrosine were assessed using immunohistochemistry.ResultsThe target vessels of cerebral cortex in 8 (72.7%) mice were occluded and the microinfarcts formed in the microinfarct group, and the average microinfarct volume was 317.23±20.29 μm3.There were remarkable neuron loss and microglia infiltration in the infarcted core, a large number of reactive astrocytes surrounding the infarcted lesion, and massive deposition of 3-nitrotyrosine in the peri-infarct area.No infarcts were observed in the sham operation group.The deposition of 3-nitrotyrosine in the sham operation group was significantly less than that in the microinfarct group (8.00±1.48 vs.98.38±9.10;t=23.962, P<0.001).Conclusions The mouse model of cerebral cortical microinfarct induced by two-photon microscopy is reliable, and its histopathologic changes are consistent with the pathologic features of cerebral microinfarct.

Objective To investigate the level of occupational well-being among the community nurses and analyze factors influencing their occupational well-being,in order to improve community nurses' well-being.Methods In January 2013,a total of 156 nurses from five community health service centers in Shanghai were selected as subjects by convenience sampling,who were surveyed with general information questionnaire and occupational well-being questionnaire.Results The overall score of occupational well-being among 156 community nurses was (161.20 ± 22.22),and the scores of all dimensions were above the medium level,among which the dimension of "interpersonal relationship" reached the highest score of (4.88 ± 0.68),but "welfare treatment" score was (3.80 ± 0.82) and the lowest.The scores concerning the two dimensions of "work value" and "self-acceptance" showed statistically significant differences among nurses who had different monthly income and years of working (F =2.845,3.360,3.056,3.164,respectively; P ＜ 0.05).Conclusions The occupational well-being of nursing staffs from community still should be improved,especially in the dimension of "welfare treatment".The managers should actively care about the nurses who have lower seniority and lower income,in order to increase their recognition of work value and self-acceptance.

Oxidative stress can induce significant cell death by apoptosis. We explore whether prior exposure to H2O2 (H2O2 preconditioning) protects PC12 cells against the apoptotic consequences of subsequent oxidative damages and what role the ATP-sensitive potassium (K(ATP)) channels play in the preconditioning protection. PC12 cells were preconditioned with 90 min exposure to H2O2 at 10 micromol/L, followed by 24-h recovery and subsequent exposures to different concentrations (20, 30, 50 and 100 micromol/L) of H2O2 for 24 h respectively. We used PI staining flow cytometry (FCM) to observe the apoptosis of PC12 cells. It was shown that 24-h exposures to H2O2 at 20, 30, 50 and 100 micromol/L respectively induced substantial cell apoptosis, which was greatly prevented in the preconditioning cells, indicating that H2O2 preconditioning protected PC12 cells against apoptosis induced by H2O2. Administration of pinacidil (10 micromol/L), an K(ATP) channel activator, significantly attenuated the apoptosis of PC12 cells induced by H2O2 at 30 and 50 micromol/L for 24 h respectively. Glybenclamide (10 micromol/L), a K(ATP) channel inhibitor, significantly suppressed or abolished the protective effects caused by the pinacidil but not by H2O2 preconditioning. However, when both H2O2 preconditioning and pinacidil were co-applied, their protection against the apoptosis of PC12 cells was much stronger than that of the individual one of them. These results suggest that H2O2 preconditioning protects PC12 cells against apoptosis and that the activation of K(ATP) channels is not involved in, but synergetically enhances adaptive protection of H2O2 preconditioning.
Animals ; Apoptosis ; drug effects ; Hydrogen Peroxide ; pharmacology ; Oxidants ; pharmacology ; Oxidative Stress ; PC12 Cells ; Potassium Channels ; metabolism ; Rats

Objective: Apolipoprotein E (ApoE) is mainly synthesized in the liver. So far, it is unknown the relationship among APOE gene polymorphisms and WML, brain atrophy. Therefore, the aim of the study was to assess the associations of APOE gene polymorphisms in patients with WML and brain atrophy. Methods: A total of 58 patients with WML, 128 patients with brain atrophy, 112 patients with co-occurrence of WML and brain atrophy and 95 healthy elderly volunteers were recruited from Renmin Hospital of WuHan University. Results: Allele E3 was the most common allele. The alleles E2 had significantly higher levels of ApoB and lower age in WML group. The alleles E2 was associated with the lower level of ApoB, LDL-Ch, TCh, and sdLDL in co-occurrence group. The E3/E3 genotype has higher level of sdLDL, but lower age and female frequency in WML. The E3/E4 genotype had higher level of TG, but lower age in WML. Gender, Age, E2, Hyperhomocysteinemia and UA were also significantly associated with disease progression. Conclusion This study found that clinical data, lipids and metabolic complications were closely related to ApoE genotypes and alleles, and also disease progression and type.

Total body irradiation combined with cyclophosphamide (TBI/CY) and busulphan combined with cyclophosphamide (BU/CY) are standard conditioning regimens in hematological stem cell transplantation for patients with myelogenous leukemia. This study was aimed to compare the therapeutic efficacy of TBI/CY and BU/CY as conditioning regiment for acute or chronic myelogenous leukemia. The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, CNKI, CBM (Chinese Bio-medicine Database) had been searched for all relevant articles (1999-2007). Comparative studies were carried out on clinical therapeutic effects of TBI/CY and BU/CY including stem cell engraftment, relapse, complications, transplant-related mortality, and disease-free survival. A meta-analysis was performed using Review Manager 4.2 software and funnel plot regression was adopted to assess the publication bias. The results indicated that 2149 articles in English and 46 articles in Chinese were got, and finally 9 clinical trials with total 3039 patients have been assessed. No significantly difference was found in engraftment failure and transplant-related mortality resulting from TBI/CY and BU/CY conditioning regimens, but the incidence of veno-occlusion of liver and hemorrhagic cystitis obviously increased in BU/CY group after transplantation, the acute GVHD, interstitial pneumonia and cataract significantly increased in TBI/CY group. The relapse rate of AML in TBI/CY group was lower than that in BU/CY group, and the rate of long-term disease-free survival of AML patients in TBI/CY group also significantly lower than that in BU/CY group, but the relapse rate of CML in TBI/CY group after transplantation was obviously higher than that in BU/CY group, but there was no difference in longterm disease-free survival rate between the two conditioning regimens mentioned above. It is concluded that the meta-analysis confirms different effects of TBI/CY and BU/CY regimens on myelogenous leukemia transplantation. This result is useful for physicians to select treatment regimens.
Busulfan ; therapeutic use ; Cyclophosphamide ; therapeutic use ; Disease-Free Survival ; Humans ; Leukemia, Myeloid ; radiotherapy ; surgery ; Leukemia, Myeloid, Acute ; radiotherapy ; surgery ; Transplantation Conditioning ; methods ; Treatment Outcome ; Whole-Body Irradiation

SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.
Cells, Cultured ; Humans ; Plasmids ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; RNA, Viral ; metabolism ; Ribonuclease III ; physiology ; SARS Virus ; genetics

In the present study, an attempt was made to prove the question whether endothelial cell precursors exist in blood circulation during postnatal period. CD34(+) cells were harvested from G-CSF mobilized adult blood and umbilical cord blood and incubated onto fibronectin/gelatin-coated Petric dishes in the presence of recombinant human vascular endothelial cell growth factor(rhVEGF) and basic fibroblast growth factor(rhbFGF). Endothelial cell lineage was identified by von Willebrand factor(vWF) expression and Ulex europous agglutinin I(UEA-I) binding capacity. The results showed that a firmly adherent cell monolayer formed when CD34(+) cells, but not CD34(-) cells, were cultured for 5 - 6 weeks as described before. Immunocytochemistry and flow cytometry analysis showed that almost all of the adherent cells were vWF-positive and around 90% were able to bind UEA-I specifically. These findings demonstrate that angioblasts exist in the circulation during postnatal life and therefore, vasculogenesis might occur in adults.

OBJECTIVETo evaluate the effect of cytomegalovirus (CMV) infection following kidney transplantation on long-term renal function and its mechanism.

METHODSNinety-six patients undergoing kidney transplantation between March 2000 and December 2005, who completed a 3-year follow-up investigation, were divided into 3 groups according CMV-pp65 antigenemia and clinical symptoms. Group A consisted of 33 recipients with symptomatic active CMV infection, group B included 33 with asymptomatic active CMV infection and group C included 30 with inactive infection. The relation of CMV infection, transforming growth factor-beta1 (TGF-beta1) mRNA in the peripheral blood mononuclear cells (PBMCs) and serum creatinine (Scr) were analyzed, and the grafts in 6 cases with renal dysfunction were biopsied.

RESULTSThe expression of TGF-beta1 mRNA in PBMCs was significantly higher in group A than in the other two groups 6 months after the transplantation (P<0.01), while Scr levels showed no significant difference between the 3 groups (P>0.05). Three years later, Scr levels in group A were significantly increased as compared with those in the other two groups (P<0.01), and the rate of renal dysfunction in group A (10/33) was significantly higher than those in group B (3/33) and C(3/30) (P<0.05). In the 16 with renal dysfunction, the expression of TGF-beta1 mRNA in PBMCs significantly higher than that in the other 80 patients with normal renal function (P<0.01). Renal allograft biopsies demonstrated mild or severe interstitial fibrosis, tubular atrophy and mononuclear cell infiltration in the 6 patients with renal graft dysfunction, supporting the diagnosis of chronic allograft nephropathy (CAN).

CONCLUSIONSymptomatic active CMV infection in renal allograft recipients is an important factor contributing to the occurrence of CAN. Monitoring of TGF-beta1 mRNA expression in PBMCs proves useful in identifying patients at risk of CAN.

Adult ; Creatinine ; blood ; Cytomegalovirus Infections ; blood ; metabolism ; physiopathology ; Female ; Humans ; Kidney ; metabolism ; physiopathology ; virology ; Kidney Transplantation ; Leukocytes, Mononuclear ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; Transplantation, Homologous