OBJECTIVE Diabetes-induced endothelial cell (EC) dysfunction and neovasculariza-tion impairment constitute vascular complications with limited treatment regimens.Transcription factor FOXO1 is a key angiogenic regulator and plays a pathologic role in progression of diabetes.The pres-ent study was designed to determine the involvement of FOXO1 in impaired EC function and post-isch-emic neovascularization in diabetes and investigate underlying mechanisms.RESULTS We found that FOXO1-selective inhibitor AS1842856 improved blood flow recovery and capillary density in ischemic hindlimb,and rescued the delay of wound closure with a concomitant augmentation of mean perfusion rate in diabetic mice. In vitro,treatment with AS1842856 or FOXO1 siRNA abrogated high glucose-in-duced apoptosis and ameliorated capillary tube formation in human umbilical vein endothelial cells(HU-VECs). FOXO1 inhibition relieved alterations in mitochondrial networks and significantly suppressed the over production of mitochondrial reactive oxygen species(mtROS)induced by high glucose in ECs. Expression of dynamin-relatedprotein-1 (Drp1) and phosphorylation at Ser616, a protein required for mitochondrial fission, were enhanced by hyperglycemia, which could be neutralized by FOXO1 inhibition. Moreover, the transcription of Rho-associated coiled-coil containing protein kinase 1 (ROCK1), which phosphorylates Drp1 at Ser616, was shown by luciferase assay to be directly regulated by FOXO1. CONCLUSION These findings suggested that FOXO1 is critical to preserve mitochondrial quantity and func-tion in ECs,and FOXO1 may serve as a therapeutic target for microvascular complications of diabetes.

The fragmentation pathways of five estrogens (estradiol, estrone, equilin sulfate, 17 a-dihydroequilin sulfate and equilenin sulfate) have been studied with high resolution and high mass accuracy using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS) in the negative ion mode. Molecular weights were obtained from [M-H](-) ions in the product ion spectra. The results indicate that the five structurally similar estrogens have similar fragmentation pathways. Using their stable isotope forms as internal reference compounds, the accurate mass and composition of the fragment ions were determined. During collision-induced dissociation (CID), cleavage is initiated by loss of oxygen atoms from carbon-17, after which D and C rings cleave sequentially and rearrange to finally form stable conjugate structures with highly abundant characteristic fragment ions at m/z 183 (accompanied by m/z 181), m/z 169 and m/z 145 (accompanied by m/z 143). Understanding these characteristic fragmentation pathways of estrogens will be helpful in identifying the structures of steroid hormones in general.
Chemical Fractionation ; methods ; Equilenin ; chemistry ; Equilin ; analogs & derivatives ; chemistry ; Estradiol ; chemistry ; Estrogens ; chemistry ; Estrone ; chemistry ; Ions ; Spectrometry, Mass, Electrospray Ionization

Objective To observe the changes in cardiopulmonary exercise test (CPET) variables of children with frequent premature ventricular contraction (PVC)before and after creatine phosphate treatment,to examine the difference of CPET results between PVC patients and healthy children,and to evaluate the effect of frequent PVC on the cardiac reserve function in children.Methods One hundred and nine frequent PVC children and 98 healthy children underwent treadmill exercise test and CPET respectively,the changes of CPET variables were observed among patients before and after treatment as well as among the healthy children.CPET variables include maximal oxygen consumption/kg (VO2max/kg),maximal oxygen consumption/heart rate( VO2max/HR),HR( at different time point),anaerobic threshold ( AT),and AT/VO2 maximum prediction (AT/VO2max pred).Results Before creatine phosphate treatment,VO2max/kg,VO2max/HR and AT/VO2max in frequent PVC children were ( 22.9±7.4 ) ml/( kg·min),( 9.3 ± 1.5 ) ml,( 15.5 ±2.7 ) ml/( kg· min).After creatine phosphate treatment,VO2 max/kg,VO2 max/HR and AT/VO2 max were (26.4 ± 6.0) ml/( kg· min),( 11.4 ± 3.3 ) ml,and ( 17.4 ± 3.8 ) ml/( kg· min).These CPET variables after creatine phosphate treatment in frequent PVC children were obviously higher than those before treatment and there was a significant difference [ VO2 max/kg ( t =2.11,P ＜ 0.001 ),VO2 max/HR ( t =4.02,P ＜ 0.001 ),AT( t =10.2,P ＜ 0.001 )].Control group of 98 healthy children had negative CPET results.Conclusion Cardiac reserve function decreases in frequent PVC children.After treatment,the exercise capacity and cardiac reserve function can be improved.CPET can reflect cardiac functional reserve changes in PVC children.

OBJECTIVETo study the role of the HBV-infected mothers' PBMC in intrauterine transmission of HBV to their fetuses.

METHODSThirty pregnant women with serum HBV DNA negative and PBMC HBV DNA positive and their newborns were used as the study group. Ten pregnant women with serum HBV negative and their infants served as the control group. HBV DNA in serum and in PBMC was detected using nested polymerase chain reaction (n-PCR). The mothers' PBMC in newborns' peripheral blood was examined using heminested-PCR.

RESULTSFour newborns were serum HBV DNA positive and 8 newborns were HBV DNA positive in PBMC in the study group. Among them, 2 newborns were HBV DNA positive in both serum and PBMC, 6 cases were positive in PBMC only, and 2 cases were positive in serum only. Five mothers had the GSTM1 gene; and it was not detected in 3 newborns. Among the 8 newborns with HBV DNA positive in PBMC, 3 did not have the GSTM1 gene, at the same time their mothers possessed the GSTM1 gene. Mothers' PBMC were detected in all of these three newborns' peripheral blood. HBV DNA in serum and in PBMC of the control group infants were all negative.

CONCLUSIONHBV-infected PBMC of the mother may serve as a vector in HBV intrauterine infection.

Adult ; DNA, Viral ; analysis ; Female ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; blood ; transmission ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; Leukocytes, Mononuclear ; virology ; Pregnancy ; Pregnancy Complications, Infectious ; virology

OBJECTIVETo construct a recombinant lentiviral vector for manganese superoxide dismutase (MnSOD) gene expression, and observe its effect on the proliferation of esophageal cancer cells in vitro.

METHODSChemical methods were employed for synthesis of the MnSOD cDNA sequence sections, along with the attB sites. Target gene fragment was constructed on the pMD-18T vector, and the recombinant plasmid pDONR221 was obtained after BP recombination reaction. Sequencing was followed by LR recombination reaction between the plasmid and DEST to obtain the lentiviral vector, which worked with helper plasmid for co-transfection of human embryonic kidney epithelial cells (293T cells). Amplification was done to determine its titer, and both transfection and selection procedures were made to get two stable transfected esophageal cancer TE-1 cell lines with medium MnSOD expression (TE-1Mm cells) and high MnSOD expression (TE-1Mh cell), and empty vector cell (TE-1Mn cells). Reverse transcription polymerase chine reaction (RT-PCR), immunofluorescence, immunocytochemistry and Western blot were used to detect the target gene with respect to its expression in the TE-1 cells. Additionally, colorimetric 3-[4,5-dimethy thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, agar colony formation assay, annexin V-FITC/PI staining and flow cytometry experiments were also conducted as to observe the influence of the medium and high MnSOD overexpressions on the proliferation of esophageal cancer cells.

RESULTSRT-PCR indicated that the transfected TE-1 cells showed positive MnSOD expression at different levels. Immunofluorescence, immunocytochemistry and Western blot suggested that TE-1Mm cells and TE-1Mh cells had MnSOD protein expression at different levels. MTT assay indicated that TE-1Mm cells had a significantly decreased survival rate compared with that of the two control cells (TE-1 cells and TE-1Mn cells), and TE-1 Mh cells had an significantly increased survival rate (P<0.05). The colony formation ability of TE-1Mm cells was (23.0 +/- 2.7)%, and that of TE-1Mh cells was (45.3 +/- 4.5)%, significantly different form the (34.7 +/- 4.2)% in TE-1 cells and (33.7 +/- 4.7)% in TE-1Mn cells (P<0.05). Annexin V-FITC/PI double staining experiment of the stably transfected cells cultured for 48 h showed that the early apoptosis rate in TE-1Mm cells was (10.6 +/- 1.0)%, significantly higher than (2.6 +/- 0.2)% in the TE-1 cells, (2.5 +/- 0.6)% in the empty vector cells and (1.0 +/- 0.1)% in the TE-1Mh cels (P<0.05). The fluorescence index (FI) of mitochondrial apoptosis of TE-1Mm cells was 0.948 +/- 0.019, significantly lower than that of TE-1 cell (1.000 +/- 0.022) and empty vector The fluorescence index of TE-1Mn cells (0.997 +/- 0.023) and TE-1 cells (1.000 +/- 0.022) were significant different from that of 0.948 +/- 0.019 in TE-1Mm cells and 1.076 +/- 0.022 in TE-1Mh cells, indicating a significant difference of mitochondrial apoptosis between the cell groups. FCM results indicated that the ROS fluorescence index of TE-1Mm cells was 0.859 +/- 0.040, that of TE-1Mh cells was 0.763 +/- 0.039, significantly lower than that of TE-1 cells (1.000 +/- 0. 042) and empty vector cells (1.002 +/- 0.047) (P<0.05).

CONCLUSIONSStably transfected cell lines with MnSOD expression have been successfully established. MnSOD overexpression shows bidirectional effect on the proliferation of esophageal cancer cells.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Mitochondria ; pathology ; Plasmids ; RNA, Messenger ; metabolism ; Reactive Oxygen Species ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Superoxide Dismutase ; genetics ; metabolism ; Transfection

Homology of three WSSV isolates, which were sampled from r epresentative maritime space of China: Tanghai isolate (Bo Bay of China), Ningbo isolate (East China Sea), Shenzhen isolate (South China Sea) was compared. Both of the genome RFLP patterns and the characteristic structural proteins SDS-PAG E electrophero grams showed that they were quite same. It suggested that they were the same ki nd of WSSV virus that caused explosive epidemic diseases of shrimps (EEDS) throu ghout southern and northern China. The same large PCR products achieved when usi ng the PCR primers from RV－PJ＝PRDV (P. japonicus, Japan) and WSBV＝PmNOBII I(P.monodon Taiwan, China) respectively to amplify the genome from P.chine nsis (Tanghai, China) with high fidelity Taq Polymerase. The sequence identiti es of WSSV from P. chinensis with those from RV－PJ＝PRDV (P.japonicus, Japan) and WSBV＝PmNOBIII (P.monodon Taiwan, China) are 97% and 100% respect ively, the results provided additional evidence that WSSV reported in different parts of the Asian and Pacific regions maybe quite the same or just different va riants of the same virus.

Objective To evaluate the efficacy and safety of memantine in the treatment of patients with Alzheimer' s disease (AD).Methods This was a 16-week,multi-center,randomized,double blind, placebo-controlled clinical trial (Study 10116).A total of 258 AD patients (MMSE score 5—18) were randomized in a 1:1 ratio into either memantine 10—20 mg/day (MEM,n=128) or placebo (PBO,n= 130) group for 16 weeks.Efficacy was primarily assessed in terms of changes of severe impairment battery (SIB) score in patients from baseline up to SIB assessment in the 16th week (16-week completers set, CS16).While ehanges of MMSE,ADCS-ADL_(19),and NPI (neuropsychiatric inventory) were evaluated as secondary efficacy parameters on both CSI6 and full-analysis set (FAS).Safety was assessed by physical examination,lab assays,ECG,and adverse events.Results 236 subjeets (CS16:MEM n=117,PBO n=119) were eligible for the efficacy assessment.No statistically significant difference between the treatments was observed on the primary and seeondary efficacy analysis,although both treatment groups had a slight increase from baseline in SIB total score.Post hoe evaluation of the data identified two bias factors that had a significant impact on the results of the pre-protoeol specified primary and secondary analyses.In a re-analysis of the data (CS16_(modified),MEM n=94,PBO n=95) excluding patient data affeeted by these factors,memantine-treated patients showed a statistically significant improvement related to placebo in the 16th week on the SIB (MEM 2.2 vs PBO 0.3,P=0.04),MMSE (MEM 1.0 vs PBO 0.1,P=0.03),and ADL (MEM 0.1 vs PBO-1.6,P=0.02) scales,indicating that memantine improved the cognitive function of AD patients and stabilized the activity of daily life.Memantine was well tolerated with an adverse event profile similar to that of placebo.Conclusion This study provides further support for pre-existing data,showing that memantine is efficacious,safe,and well-tolerated in patients with moderate to severe AD.

Chronic osteomyelitis is a common disease. Traditionally, osteomyelitis is treated with parenteral antibiotics for 4 to 6 weeks after thorough debridement. However, this antibiotic treatment course has no documented superiority over other courses of antibiotic treatment. As this disease has been understood more deeply and various treatment technologies have been explored recently, the use of antibiotics has also changed. This article reviews the latest progress concerning the course of antibiotic treatment for chronic osteomyelitis.

BACKGROUNDRight ventricular function plays an important role in the hemodynamic derangement during off-pump coronary artery bypass (OPCAB) surgery. Pressure-volume loops have been shown to provide load-independent information of cardiac function. Therefore, the aim of this study was to investigate the feasibility of construction of right ventricular pressure-volume loops with pressure and volume data measured by a volumetric pulmonary artery catheter (PAC) and to evaluate right ventricular systolic and diastolic function by end-systolic elastance (E(ES)) and end-diastolic stiffness (E(ED)) in OPCAB surgery.

METHODSTwenty-eight patients who underwent OPCAB surgery were included. After anesthesia induction, a volumetric PAC was placed via the right internal jugular vein. Data were recorded at: anesthesia steady-state before skin incision (T1); 5 minutes after the stabilizer device was placed for anastomosis on the heart's anterior wall (T2), lateral wall (T3), posterior wall (T4), respectively; after sternal closure (T5). Three sets of data were collected at each time point: first, hemodynamic variables were measured; second, right ventricular E(ES) and E(ED) were calculated; third, right ventricular pressure-volume loops were constructed with pressure and volume data measured from end-diastole point, end-isovolumic systole point, peak-ejection point, end-systole point and end-isovolumic diastole point.

RESULTSRight ventricular pressure-volume loops generally shifted to the left during OPCAB surgery. Especially, the end-diastolic point shifted upward and to the left at T2-T5 compared with that at T1. Decrease in right ventricular ejection fraction, stroke volume index and end-diastolic volume index occurred (P < 0.05) at T4 compared with values at T1. Pulmonary vascular resistance index at T4 increased relatively compared with that at T2 and T3. The change of E(ES) was not statistically significant during operation. Right atrial pressure increased only during coronary anastomoses (T2-T4, P < 0.05), whereas E(ED) increased throughout OPCAB surgery (P < 0.05).

CONCLUSIONSRight ventricular pressure-volume loops can be constructed using a volumetric PAC. Right ventricular systolic dysfunction occurred during anastomoses on the heart's posterior wall not due to impaired myocardial contractility but as a result of reduced preload and a relative increase in afterload. Right ventricular diastolic function was impaired throughout OPCAB surgery.

Aged ; Blood Pressure ; Coronary Artery Bypass, Off-Pump ; methods ; Female ; Hemodynamics ; Humans ; Male ; Middle Aged ; Ventricular Function, Right ; physiology

OBJECTIVETo compare the immunophenotypic and clinical characteristics between NPM1 mutated acute myeloid leukemia (AML) (NPM1m(+)AML) and unmutated AML(NPM1m(-)AML) not otherwise characterized (NOS) under similar FAB subtypes constituent ratio.

METHODSImmunophenotyping and NPM1 gene mutation type-A, B and D and other leukemic related fusion genes were detected by multiparameter flow cytometry and real time RT-PCR or PCR, respectively. 104 AML patients with NPM1m(+)AML and performed immunophenotyping assay were included, 97 with NPM1m(-)AML.

RESULTSThere were significant difference between the two groups at presentation in terms of sex, white blood count(WBC), platelet counts (PLT), blast ratio, normal karyotype ratio, WT1 expression level, FLT3-ITD mutation positive rate and remission rate of first course of induction therapy (P < 0.05). On the immunophenotype, the expression of early differentiation antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2), myeloid and monocytic differentiation-associated antigens (CD13, CD14, CD15) were lower, and that of CD33 as well as CD123 were higher in NPM1m(+)AML patients. Among them, only CD34, HLA-DR, CD7, and CD4 positive cases were significantly lower in NPM1m(+)AML group than in NPM1m(-)AML group (P < 0.05), the rest of them had significant difference in the number of positive cells (P < 0.05). Above features were further analyzed between the M1/M2 and M4/M5 subgroups. M1/M2 cases retained the women prominent and had a higher WT1 expression level (P < 0.05). The expression of monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens were higher and that of CD117 were lower in M4/M5 subtype (P < 0.05). Among them, the positive rates of HLA-DR, CD64, CD11b, CD10, CD15, and CD4 were significantly higher in M4/M5 than in M1/M2 in NPM1m(+)AML group (P < 0.05).

CONCLUSIONThe most clinical characteristics in NPM1m(+)AML patients are consistent with reports, but some immunophenotype are different to the previous reports under similar FAB subtypes constituent ratio. The major immunophenotypic features of NPM1m(+)AML patients are lower expression of progenitor, myeloid and lymphoid lineage antigens. Monocytic differentiation-associated antigens are only higher expression in M4/M5 cases when comparison with M1/M2 cases within NPM1m(+)AML group.

Adolescent ; Adult ; Aged ; Antigens, CD ; metabolism ; Child ; Child, Preschool ; Female ; HLA-DR Antigens ; immunology ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; immunology ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Young Adult