OBJECTIVETo establish fluorescent quantitative PCR method for detecting human herpes virus type 6 (HHV6).

METHODSAccording to the specific sequence of human herpes virus type 6 genes, the primers and the fluorescent probe (TaqMan) were designed and synthesized. The fragment generated from HHV 6 gene as template was cloned into the pMD18-T vector which was constructed from the pUC 18. And the positive recombinant plasmid was 1:10 diluted and used as standard quantitative template to make the standard curve for sample detection.

RESULTSThe standard curve indicated the linear relationship between Ct (cycle threshold) and template concentration. The clinical samples from 135 cases were detected by this system, 16 cases among 135 were positive.

CONCLUSIONThe fluorescent quantitative PCR method for the detection of human herpes virus type 6 is simple and accurate, and this method may be helpful to clinical diagnosis.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; DNA Primers ; DNA Probes ; DNA, Viral ; genetics ; Female ; Herpesvirus 6, Human ; genetics ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Purpura, Thrombocytopenic, Idiopathic ; diagnosis ; virology ; Reproducibility of Results ; Roseolovirus Infections ; diagnosis ; virology ; Sensitivity and Specificity ; Young Adult

OBJECTIVETo establish a quantitative method for calceolarioside B in Caulis Akebiae.

METHODThe samples were separated at 35 degrees C on a Phenomenex Luna C18 column eluted with methanol-water-phosphoric acid (35: 65: 0.05) as mobile phase. Flow rate was at 1.0 mL x min(-1) and the detection wavelength was at 330 nm.

RESULTThe calibration curve was linear over the range from 0. 04 to 0. 86 g (r = 0. 9998) and the average recovery was 97.2%. 39 Crude drug materials collected from different areas were determined and the contents of calceolarioside B in Caulis Akebiae were fluctuated from 0. 021% to 0. 745%.

CONCLUSIONThe method is simple and repeatable and could be used for the quality control of Caulis Akebiae.

Caffeic Acids ; analysis ; chemistry ; China ; Chromatography, High Pressure Liquid ; methods ; Ecosystem ; Glucosides ; analysis ; chemistry ; Magnoliopsida ; chemistry ; classification ; growth & development ; Plant Leaves ; chemistry ; Plant Roots ; chemistry ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; classification ; growth & development ; Reproducibility of Results

To investigate the non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line, a recombinant plasmid vector, pRC/RSV-hFVIIIBDcDNA, was constructed by cloning B-domain-deleted (Delta760aa-1639aa) human factor VIII cDNA (hFVIIIBDcDNA) into plasmid vector, pRC/RSV. The plasmid RC/RSV-hFVIIIBDcDNA was then transfected by means of SuperFect Transfection Reagent into mouse 32D cell line. After screening with G418, the procoagulant activity (hFVIII:C) and antigen (hFVIII:Ag) of human factor VIII in the culture medium were detected using one-stage method and ELISA, respectively. Furthermore, RT-PCR was performed to observe the transcription of hFVIIIBDcDNA. The results showed that human coagulation factor VIII protein existed in culture medium with hFVIII:C up to 2.01 U/(10(6) cell x 24 hours) and hFVIII:Ag to 450.08 ng/(10(6) cell x 24 hours). RT-PCR displayed mRNA of hFVIIIBDcDNA in 32D cells. It is concluded that the recombinant plasmid RC/RSV-hFVIIIBDcDNA can successfully express human FVIII in mouse 32D cell line, and hFVIII expressed in vitro presents the similar coagulant activity to the native hFVIII existing in normal human plasma.
Animals ; Cell Line ; DNA, Complementary ; genetics ; Enzyme-Linked Immunosorbent Assay ; Factor VIII ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Mice ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo assess the value of apparent diffusion coefficient (ADC) of magnetic resonance diffusion-weighted imaging (MR-DWI) for diagnosis of the liver pathologies in rabbit model of liver fibrosis.

METHODMR-DWI with four different b values (200, 500, 300 and 600 s/mm(2)) was performed in 4 normal New Zealand white rabbits and 13 rabbits with experimental liver fibrosis. For each rabbit, 4 ADC values were obtained in the left and right lobes of the liver. According to the ISHAK criteria of liver histopathological scoring and fibrosis staging system, all the liver specimens were histopathologically graded (scores 1-6 for grade I, 7-12 for grade II, and 13-18 for grade III) and assessed for fibrosis staging (stages I to VI). The variation of ADC values were analyzed based on the results of histopathological grading and fibrosis staging.

RESULTSThe 4 ADC values were obviously lower in rabbits with liver fibrosis than in the normal control rabbits. Statistical analysis showed significant differences in the ADC values between the normal control and liver fibrosis groups, and between the rabbits with different histopathological grades and fibrosis stages (P=0.000).

CONCLUSIONSLiver fibrosis results in significantly lowered ADC values of the liver depending on the histopathological grades and fibrosis stages. The pathological basis for these changes lies in reduced water content and restricted Brownian motion of water in the liver due to hepatocyte degeneration and swelling, inflammatory cell infiltration, and collagen fiber deposition in the interstitial space.

Animals ; Diffusion ; Disease Models, Animal ; Female ; Liver ; pathology ; Liver Cirrhosis ; diagnosis ; pathology ; Magnetic Resonance Imaging ; Male ; Rabbits

OBJECTIVETo investigate the value of intensity modulated radiation therapy (IMRT) for patient with gynecological malignancies after treatment of hysterectomy and chemotherapy/radiotherapy.

METHODSAll 32 patients with cervical or endometrial cancer after hysterectomy received full course IMRT after 1 to 3 cycles of chemotherapy (Karnofsky performance status(KPS) > or =70). Seventeen of these patients underwent postoperative preventive irradiation and the other 15 patients were pelvic wall recurrence and/or retroperitoneal lymph node metastasis, though postoperative radiotherapy and/or chemotherapy had been given after operation.

RESULTSThe median dose delivered to the PTV was 56.8 Gy for preventive irradiation, and 60.6 Gy for pelvic wall recurrence or retroperioneal lymph node metastasis irradiation. It was required that 90% of iso-dose curve could covere more than 99% of GTV. However, The mean dose irradiated to small intestine, bladder, rectum, kidney and spinal cord was 21.3 Gy, 37.8 Gy, 35.3 Gy, 8.5 Gy, 22.1 Gy, respectively. Fourteen patients presented grade I (11 patients) or II (3 patients) digestive tract side-effects, Five patients developed grade I or II bone marrow depression. Twelve patients had grade I skin reaction. The overall 1-year survival rate was 100%. The 2- and 3- year survival rate for preventive irradiation were both 100%, but which was 5/7 and 3/6 for the patients with pelvic wall recurrence or retroperioneal lymph node metastasis.

CONCLUSIONIntensity modulated radiation therapy can provide a better dose distribution than traditional radiotherapy for both prevention and pelvic wall recurrence or retroperioneal lymph node metastasis. The toxicity is tolerable. The adjacent organs at risk can well be protected.

Adult ; Aged ; Combined Modality Therapy ; Diarrhea ; etiology ; Endometrial Neoplasms ; drug therapy ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Hysterectomy ; methods ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Recurrence, Local ; Radiotherapy Dosage ; Radiotherapy, Intensity-Modulated ; adverse effects ; methods ; Survival Analysis ; Uterine Cervical Neoplasms ; drug therapy ; radiotherapy ; surgery

Objective To investigate epidemic characteristics of a family cluster of COVID-19, and to provide reference in improving the criteria for exclusion diagnosis and medical observation of close contacts. Methods Field epidemiological method was used to investigate the cases and close contacts of a family cluster of COVID-19 in Pudong New Area.Descriptive analysis was conducted on epidemiological data.Real-time fluorescence quantitative RT-PCR was used to detect 2019-nCoV nucleic acid in the respiratory tract specimens. Results There were two confirmed cases and one suspected case in the family cluster.The source of infection was Case 1 with a living history in Wuhan, Hubei Province.Case 2 and Case 3, as close contacts, received 14-day medical observation in a centralized isolation site.Case 2 showed symptoms 4 days after the onset of Case 1, and the diagnosis of COVID-19 was excluded after two negative nucleic acid tests during the isolation period.However, after the expiration of isolation, Case 2 was diagnosed positively for COVID-19 and Case 3 was suspected first and then excluded. Conclusion Daily close contact is critical for COVID-19 transmission and is the major cause of family clustering.Once the close contacts show symptoms, diagnosis should be made by combining the results of nucleic acid test, chest CT test, serological test, etc.We suggest to grade the risk of infection for close contacts, and to strengthen the standard of medical observation for close contacts with high risk of infection.

Objective To determine the mass concentration of atorvasta-tin and its metabolites in human plasma by HPLC -MS/MS method. Methods The solid-phase extraction was adopted to process the blood sample, and the XTerra ? MS C18 column was used, with 95%acetonitrile-5 mmol? L-1 ammonium bicarbonate as the mobile phase, gradient elution with positive ion scan and multiple reaction monitoring mode mass concentration of the sample was measured.The specificity, standard curve and lower limit of quantification, accuracy and recovery rate, matrix effect and stability were investigated.Results The standard curve of atorvastatin and its metabolites were Y =1.82 X-2.94 ×10 -3 (r=0.999 5),Y=0.28X -4.14 ×10 -5 (r =0.999 7),Y=0.60X +1.11 ×10 -3 ( r=0.997 4 ) , and they showed a good linearship between 0.1 and 40.0 ng? mL-1 .The lowest concentration was 0.1 ng? mL-1 . The intra and inter-day RSD were less than 15%, the average recovery was higher than 80%, the stability was good.Conclusion The method is simple, rapid, sensitive and accurate and specific, suitable for phar-macokinetics study of atorvastatin and metabolites dynamics in vivo .

Objective To develop a simple and highly sensitive liquid chromatography-tandem quadrupole mass spectrometry method for deter-mination of estradiol in human plasma.Method Estradiol was extrac-ted using liquid-liquid extraction method from human plasma, with est-rodial-d3 as an internal standard.After extracted, samples were deri-vatizated by dansyl chloride in alkaline conditions.The Atlantis T3 (2.1 mm ×100 mm, 5.0 μm) column was used for separation.The gradient mobile phase was 0.1% formic acid and acetonitrile with a flow rate of 0.5 mL? min-1 , the temperature was set at 30 ℃, and the injection volume was 10 μL.The detection was performed with multiple reactions monitoring using positive electrospray ionization with API5500 as the detec-tor.Results The linear ranges of estradiol was 1 -100 pg? mL-1 , lower limit of quantification was 1 pg? mL-1 , the regression equation was y=5.47 ×10 -2 x+4.67 ×10 -2 .The inter-and intra-day RSD were less than 15%and the absolute recovers were above 97%.Estrodiol was proved to be stable under study station.Conclusion This method is specific, sensitive, accurate, and suitable for the pharmacokinetic study of estradiol in human plasma.

BACKGROUNDBlood transfusion has been found to be a devastating factor for outcomes of hepatectomy. This study was to assess the value of major hepatectomy without blood transfusion.

METHODSWe retrospectively studied 51 patients who had undergone major hepatectomy without blood transfusion, including 29 patients with primary liver cancer, from August 1997 to December 2000. Sixty patients undergoing major hepatectomy with blood transfusion including 48 patients with primary liver cancer served as controls. Hepatectomy was performed through normothermic interruption of the porta hepatis. Intraoperative ultrasonography was performed to define tumor margins, and an ultrasound dissector was used to dissect liver parenchyma.

RESULTSIn the study group, the operative mortality and morbidity and 1-, 2-, and 3-year recurrence rates were 0%, 9.8%, 24.1%, 27.6% and 31.0%, respectively. In the control group, they were 3.3%, 28.3%, 43.5%, 54.3% and 58.7%, respectively. Significant differences were seen in morbidity and recurrence rates of patients with liver cancer between the two groups (P < 0.05).

CONCLUSIONMajor hepatectomy without blood transfusion can reduce postoperative morbidity and recurrence rate of patients with liver cancer.

Adult ; Aged ; Blood Transfusion ; Female ; Hepatectomy ; methods ; mortality ; Humans ; Liver Neoplasms ; mortality ; pathology ; surgery ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Postoperative Complications ; prevention & control

OBJECTIVETo explore the gene mutation type of an inherited coagulation factor VII deficiency pedigree.

METHODSFVII:Ag, FVII:C, FVIIa were detected to classify deficiency type. FVII gene mutations were analysed in the proband and her family members by DNA directly sequencing. Biostructural pathology of the identified mutation was analysed by molecular modeling.

RESULTSHomozygosity of C-->T transition at position 11514 in exon 8 resulting in Thr359Met was identified in the proband, and heterozygosity for Thr359Met was confirmed in her parents, her son and some other family members. Thr359Met induces CRM-deficiency. It is found by computer simulated molecular model that the replacement of Thr by Met which has a larger and longer side chain might cause steric hindrance, and change the number of H-bonds.

CONCLUSIONSHomozygous missense mutation Thr359Met was found in a pedigree of hereditary FVII deficiency. This mutation might change the configuration of protein molecule and result in severe FVII deficiency.

Adolescent ; Adult ; Aged ; DNA Mutational Analysis ; Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Homozygote ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Pedigree ; Polymerase Chain Reaction