A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.
Animals ; Antibodies, Monoclonal ; Antibodies, Neutralizing ; Blotting, Western ; Capsid Proteins ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Genotype ; Hepatitis E virus ; Mice ; Mutant Proteins ; biosynthesis ; Open Reading Frames ; Recombinant Proteins ; biosynthesis

Objective To investigate the clinical features,diagnosis and treatment experience of idiopathic calcinosis of the scrotum(ICS).Methods We report 5 cases with a 4-month to 20-year history of ICS.The related literatures were reviewed.Results The multiple scrotum masses in these patients were increased slowly in size and number.The levels of blood calcium and phosphorus were normal.Histopathologically,nodules were composed of basophilic calcified material and located in the dermis.There was no epithelial lining around the calcified nodules.Conclusion ICS is a rare localized benign disease.The diagnosis of the tumor relies largely on the histopathology.Surgical excision is the best choice for treatment.

BACKGROUND:Donor selection for high-risk acute leukemia is still controversial.OBJECTIVE:To compare the therapeutic efficacy of the unrelated donor peripheral blood and matched sibling allogeneic hematopoietic stem cell transplantation for high-risk acute leukemia.METHODS:Total 65 patients with high-risk acute leukemia treated during January 2008 to January 2016 were included,in which 30 patients chose the unrelated donor peripheral blood stem cell transplantation (UD), and other 35 chose the matched sibling allogeneic hematopoietic stem cell transplantation (MS) according to the wishes of patients and their own situation. After treatment, the chi-square test, Kaplan-Meier survival analysis method, and other methods were used to compare the implanted and hematopoietic reconstitution, the occurrence of graft-versus-host disease, relapse mortality and long-term survival between the two groups.RESULTS AND CONCLUSION:The implantation rate, platelet hematopoietic reconstitution time, the incidence of acute and chronic graft-versus-host disease, and its type exhibited no significant differences between the two groups (P > 0.05).The relapse rate, total death rate, and transplant-related mortality rates were 10.0%, 50.0%, and 40.0% in the UD group and 20.0%, 48.6%, and 25.7% in the MS groups, respectively, and the intergroup difference was insignificant (P > 0.05).The expected 2-year cumulative disease-free free survival and overall survival rates were (49.4±9.2)% and (52.6±9.2)% in the UD group and (53.9±8.5)% and (53.9±8.5)% in the MS group, respectively, and the intergroup difference was also insignificant (P > 0.05). Our experimental findings show that unrelated donor peripheral blood stem cell transplantation can be used as an effective alternative in the absence of sibling donors.

Objective To assess the consistency of four standardized cystatin C particle-enhanced turbidimetric assay (PETIA) and one particle-enhanced nephelometric immunoassay (PENIA) measurement systems Methods Performance verification test was conducted according to CLSI EP 15-A2 and EP9-A2. Fourty serum samples in comparative test were obtained from the remaining serum samples of outpatients in Peking Union Medical College Hospital in March 2013.Fourty serum samples were tested on Olympus AU5400 automatic biochemical analyzer ( four PETIA Cys C reagents:Shanghai Jingyuan Co ., Ltd, Beijing Leadman Biochemistry Co ., Ltd, Beijing Strong Biotechnologies , Maker Biotechnology in Sichuan , and labelled as A, B, C, D respectively) and PENIA N Latex Cys C measurement system on Siemens BNⅡ(labelled as E).Correlation analysis were performed among four PETIA methods one PENIA method Differences of each detection system were compared in the medical decision level 1,2,3,4 mg/L.The reference material ERM-DA471/IFCC was measured by five systems and bias ( percentage bias ) was calculate for each system.Results Results of systems A, B, C, D, E were 1.29(0.89-2.43), 1.30 (0.96-2.59), 1.22(0.90-2.44), 1.27(0.96-2.47), 1.14(0.82-2.05)mg/L.Chart shows bias among these five systems was small when Cys C concentration was less than 4mg/L.PETIA method A, B, C, D correlated with their mean value well , with the average deviation from their mean value ( percent deviation) at -0.017 -0.031 mg/L ( -3.1%-2.1%), and all were less than allowed bias from the biological variation (3.4%).The deviation of PETIA method A, B, C, D with their mean value in medical decision level at -0.176 -0.178 mg/L.Systems A, B, C, D correlated well with the result of PENIA method system E , and the mean deviation ( percent deviation ) was at 0.278 -0.326 mg/L ( 12.6%-18.5%) , and the deviation ( percent deviation ) in the medical decision level 0.055 -1.079 mg/L (5.51%-26.98%).Bias of PETIA method A, B, C, D Cys C system measuringERM -DA471/IFCC ranged from 0.22 to 0.39 mg/L ( 3.9%-7.0%) , which exceeded the allowable range of the reference material target value, and were larger than the allowable bias from biological variation (3.4%).Bias ( percent ) of PENIA method system E was -0.1 mg/L ( -1.7%) , within the allowable range of ERM-DA471/IFCC target value .Conclusions The consistency of four assesed PETIA Cys C reagents was relatively ideal, and improved markably after being traced to ERM-DA471/IFCC.Besides, the results of PETIA were higher than those of PENIA .Bias among these five systems was small when Cys C concentration was less than 4 mg/L, and the bias became larger in higher Cys C concentration.

Objective To compare the efficacy and safety of 12 weeks-entecavir (ETV) treatment in HBeAg-negative chronic hepatitis B patients with acute-on-chronic liver failure (HBV-ACLF) in comparison to lamivudine (LAM).Methods Ninety eight HBeAg-negative patients with HBV-ACLF who were nucleos(t) ide analogs treatment naive as well as with detectable serum HBV DNA were randomly divided into ETV group and LAM group,and each have 49 patients.Additional to the comprehensively basic internal medicine treatment,antiviral therapy with ETV (0.5 mg,qd) or LAM (0.1,qd) was performed,respectively.The differences of mortality rates,clinical improvement rates,complete virological response (CVR) rates and,adverse events between ETV group and LAM group were compared after 12 weeks of treatment.Results The baseline characteristics of patients in ETV group were comparable to those in LAM group.At week 12,a lower mortality rate of 28.6％ than that of LAM group (48.9％) was observed in ETV group (P ＜ 0.05).In terms of clinical improvement rate,ETV group indicated a higher tendency in total (65.3％ vs 48.3％,P =0.067) and a statistically significant value in subpopulation of model for end-stage liver disease scored no more than 30 than LAM group (75.6％ vs 52.5％,P ＜ 0.05).As expected,ETV group achieved a higher CVR rate than LAM group at week 12 (94.3％ vs 72％,P ＜0.05).Discontinuation of antiviral therapy occurred to none of the patients in both groups.Conclusions Comparing with LAM,ETV can safely inhibit HBV replication more intensively and reduce the 12 weeks mortality rate in HBeAg-negative patients with HBV-ACLF.

Artemisinin, a new and a very potent antimalarial drug, is produced by the Chinese medicinal herb Artemisia annua L. It is a sesquiterpene lactone with an endoperoxide bridge and is active against chloroquine resistant forms of Plasmodium falciparum. The relatively low yield (0.01% - 0.6%) of artemisinin in A. annua is a serious limitation to the commercialization of the drug. Therefore, a through understanding of the biosynthetic pathway and the characterization of the involved enzymes are important for the biology production of artemisinin. This review is focused on the recent progress in the molecular regulation of artemisinin biosynthesis from the following aspects: the biosynthetic pathway of artemisinin, the key enzymes involved in artemisinin biosynthesis, and the molecular regulation of artemisinin biosynthesis. The biosynthetic pathway of artemisinin belongs to the isoprenoid metabolite pathway, the key enzymes involved in the biosynthesis of artemisinin include: 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FDPS), and amorpha-4, 11-diene synthase, of which amorpha-4, 11-diene synthase catalyzes the cyclisation of the ubiquitous precursor farnesyl diphosphate to the highly specific olefinic sesquiter-pene skeletons and has been postulated as the regulatory step in the biosynthesis of artemisinin. Recently the gene encoding of the amorpha-4, 11-diene synthase has been cloned and the functional expressions have been studied by several research teams, therefore, the breakthroughs in production of artemisinin could hopefully be achieved by metabolic engineering of the plant, in particular, by over-expressing enzyme(s) catalyzing the rate limiting step(s) of artemisinin biosynthesis or by inhibiting the enzyme(s) of other pathway competing for its precursors. Besides, the effects of the heterogenesis isoprenoid pathway related genes on artemisinin biosynthesis of the transformed plants were also discussed.
Alkyl and Aryl Transferases ; genetics ; metabolism ; Antimalarials ; metabolism ; Artemisia annua ; enzymology ; genetics ; metabolism ; Artemisinins ; metabolism ; Biotechnology ; methods ; Models, Biological ; Signal Transduction ; genetics ; physiology

This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells (BCSCs). BCSCs were enriched by using serum-free medium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and flow cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a promising therapeutical approach for breast cancer.

OBJECTIVETo investigate the prognostic predictors of nasal NK/T cell lymphoma.

METHODSThe clinicopathologic feature data of 61 patients with nasal NK/T cell lymphoma proven by pathological examination from Jan. 1997 to Jan. 2005 were collected. Expression of survivin, CD44, nm23, p53, Ki-67, MDR-1 and CD95 was detected by immunohistochemical staining in 30 patients with available histologic specimens. The correlation between these factors and prognosis were analyzed.

RESULTSIn univariate analysis, performance status, LDH level, clinical stage, initial treatment response, CD56, Ki-67 and CD95 were found to be the prognostic factors associated with time to progression (TTP) in nasal NK/T cell lymphoma, while the performance status, B symptoms, LDH level, initial treatment response, Ki-67 and CD95 were demonstrated as prognostic factors related to overall survival. In multivariate analysis, clinical stage, initial treatment response and performance status were independent prognostic factors for TTP, while the latter two factors were independent prognostic factors of overall survival.

CONCLUSIONClinical stage and initial treatment response, and performance status are found to be independent prognostic factors for TTP, whereas the latter two factors are demonstrated as independent prognostic factors of the overall survival. Overexpression of Ki-67 may be an unfavorable prognostic factor, but overexpression of CD95 may be a favorable one.

Adolescent ; Adult ; Aged ; Analysis of Variance ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Biomarkers, Tumor ; analysis ; Cyclophosphamide ; therapeutic use ; Doxorubicin ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Hyaluronan Receptors ; analysis ; Immunohistochemistry ; statistics & numerical data ; Inhibitor of Apoptosis Proteins ; Ki-67 Antigen ; analysis ; Killer Cells, Natural ; drug effects ; metabolism ; pathology ; Lymphoma, T-Cell ; drug therapy ; metabolism ; pathology ; Male ; Microtubule-Associated Proteins ; analysis ; Middle Aged ; Neoplasm Proteins ; analysis ; Neoplasm Staging ; Nose Neoplasms ; drug therapy ; metabolism ; pathology ; Prednisone ; therapeutic use ; Prognosis ; Proportional Hazards Models ; Vincristine ; therapeutic use ; fas Receptor ; analysis

OBJECTIVETo modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference.

METHODSThe U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference.

RESULTSThe sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection.

CONCLUSIONThe siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Polymerase Chain Reaction ; RNA, Small Interfering ; biosynthesis ; genetics ; RNA, Small Nuclear ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo explore clinical effects of Ilizarov technique at stage I for repairing tibial post-traumatic osteomyelitis with bone and skin defect.

METHODSFrom June 2010 to December 2013,44 patients with tibial post-traumatic osteomyelitis with bone and skin defect were treated with Ilizarov technique at stage I . Among them, there were 35 males and 9 females aged from 18 to 70 years old with an average of 42.5 years old. Bone defect ranged from 4 to 16 cm, skin defect ranged from 3 cm x 4 cm to 5 cm x 16 cm. The operation was performed debridement thoroughly, removed inflammatory bone section, osteotomy invasively, install circular external fixator by Ilizarow technique; screw nut were rotated at 1 week after operation, and prolonged 0.5 to 1.0 mm everyday. Wound surface, new born callus and bone healing were observed to evaluate clinical effects.

RESULTSAll patients were followed up from 11 to 36 months with an average of 18.5 months. Bone defect after osteotomy was from 6 to 22 cm with an average of 11.5 cm; the time of wound healing time ranged from 21 to 79 d with an average of 38 d; bone defect healing time was from 8 to 15 months with an average of 12.5 months. All patients were cured, no recurrent infection, refracture and shorten of calf deformity were occurred.

CONCLUSIONRepairing tibial post-traumatic osteomyelitis with bone and skin defect by llizarov technique at stage I has advantages of less trauma, low inflammatory recurrence rate, could avoid multiple complex operation, and receive definite curative effect.

Adolescent ; Adult ; Aged ; Female ; Humans ; Ilizarov Technique ; Male ; Middle Aged ; Osteomyelitis ; surgery ; Osteotomy ; Tibia ; surgery