Protein phosphatase 4 catalytic subunit (PP4C) increases hepatitis B virus X protein levels and promotes its biological functions / 中华微生物学和免疫学杂志

Objective:To investigate the role of protein phosphatase 4 catalytic subunit (PP4C) in regulating hepatitis B virus X protein (HBx) levels and its effects on the biological functions of HBx, thus to provide a potential therapeutic targets for hepatitis B virus (HBV)-related hepatocellular carcinoma.Methods:In vivo and in vitro interactions between HBx and PP4C were analyzed by co-immunoprecipitation (Co-IP) and GST pull-down assay. Recombinant plasmids of PP4C and HBx were co-transfected with Lipofectamine 3000 reagents into hepatoma cells to detect the protein levels of HBx by Western blot. The half-life of HBx in the transfected cells treated with cycloheximide (CHX) were detected. The phosphorylation assay was used to evaluate the effects of PP4C on HBx phosphorylation. CCK8 assay, wound healing assay and Matrigel invasion chamber assay were used to analyze the effects of PP4C on the biological functions of HBx. Results:PP4C interacted with HBx in vivo and in vitro. PP4C overexpression significantly increased the protein level and stability of HBx and the phosphorylation assay confirmed that PP4C overexpression decreased the serine phosphorylation of HBx in hepatoma cells. PP4C overexpression enhanced the migration and invasion of hepatoma cells, but had no significant effects on the proliferation. Conclusions:The interactions between HBx and PP4C promoted the stability of HBx and ultimately enhanced the migration and invasion of hepatoma cells, and the mechanisms might be related to the decrease of HBx serine phosphorylation by PP4C. This study provided a theoretical basis for further investigation of the pathogenic mechanisms of HBx, and targeting PP4C and HBx interaction might provide insights for developing novel treatment for HBV-related hepatocellular carcinoma.

Genetic analysis of a pedigree affected with inherited thrombocytopenia caused by a novel mutation of MYH9 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study genetic mutations and clinical features of a pedigree affected with MYH9-related disorders from Guangxi.</p><p><b>METHODS</b>Blood platelets were counted with a hemocytometer. Blood smear was carried out to detect the inclusion body in peripheral blood neutrophils. DNA and mRNA samples were extracted from blood samples from the members of the pedigree. Fragments of the MYH9 gene were amplified with PCR and directly sequenced.</p><p><b>RESULTS</b>The affected individuals presented with a triad of giant platelets, decreased platelet count and inclusion bodies in the neutrophils with variable expressivity. A heterozygous deletional mutation (c.5803delG) in exon 41 of the MYH9 gene was found in all of the 8 affected individuals, which led to a frame-shift and change of 26 amino acids at the C-end of the tail domain of nonmuscle myosin heavy chain IIA (NMMHC-IIA) (p.Ala1935Profs*12). The same mutation was not found among healthy members of the pedigree.</p><p><b>CONCLUSION</b>The c.5803delG mutation probably underlies the MYH9-related disorders in this pedigree. The mutation has altered the C-end of the tail domain of the NMMHC-IIA protein, resulting in mild clinical symptoms in the affected individuals.</p>

Efficacy of a 980-nm laser percutaneously delivered through an optical fiber for the treatment of 167 cases of vascular malformation: a clinical observation / 中华皮肤科杂志

Objective To evaluate the efficacy and safety of endoscopy-assisted percutaneous interventional procedure with a 980-nm laser for the treatment of venous malformations.Methods A total of 167 patients with venous malformation were enrolled,including 102 females and 65 males,whose age ranged from 0.5 to 60 years.Skin lesions occurred on the limbs in 66 patients,on the head and face in 48,on the chest and abdomen in 24,on the back in 13,on the neck in 10,as well as on the scrotum in 6.A 980-nm laser operated at pulse width of 0 ms-2.5 s and pulse energy of 0-20 W with a 400 μm-diameter optical fiber.Puncture was performed after circular infiltration anesthesia around skin lesions,and then the optical fiber was inserted at the same puncture point to directly or indirectly destruct the vascular wall by photothermal effects and thermal coagulation.For deep lesions around important anatomical structures,the interventional therapy was performed with the assistance of endoscopy or ultrasound.According to the size and shape of tumors,these tumors were irradiated back and forth with speeds of 2 cm/s-4 cm/s.The optical fiber was also inserted in different directions in a fan-shaped pattern for 3-5 sessions until tumors were shrunken or even disappeared.The time interval between two treatments was 3 months,and all the patients were followed up for 4 months on average.Results Of the 167 patients,148 (88.6％)were cured,11 (6.6％) markedly improved,4(2.4％) improved,and 4(2.4％) unimproved.The response rate was 97.6％.Totally,159 patients were satisfied with the therapeutic effects after 1 session of treatment,and 8 were improved after 2 sessions of treatment with a time interval of 3 months.In addition,mild postoperative pigmentation occurred in 6 (3.6％) cases,and skin burns in 3 (1.8％) cases.After 3 months,the above symptoms were improved significantly.Two (1.2％) patients had vascular recanalization and residual lesions,but they were improved after reoperation.No adverse reactions were observed,such as bleeding,scar,tendon and nerve injuries,and dysfunction.Conclusion A 980-nm laser percutaneously delivered through an optical fiber is safe and effective for the treatment of venous malformation.

Primary cutaneous diffuse large B-cell lymphoma, leg type: a study of clinicopathology, immunophenotype and gene rearrangement / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL).</p><p><b>METHODS</b>Seven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study.</p><p><b>RESULTS</b>All the seven cases were male, and the median age was 72 years. Patients usually presented with multiple purple tumors, nodules, papules and infiltrative plaques. Two patients had a history of leg injury before onset, and one had mosquito bites. Histologically, the tumor involved the dermis and subcutis with dense and diffuse infiltrative pattern composing of centroblasts and/or immunoblasts. Immunohistochemical staining showed that seven cases (7/7) expressed CD20, six (6/6) expressed bcl-2, four (4/4) expressed MUM-1, four (4/5) expressed CD79a, four (4/5) expressed PAX-5 and four (4/6) expressed bcl-6, respectively. All cases did not express CD3ε, CD45RO, CD10 and CD30. IgH gene rearranged bands were detected in three (3/6) cases and Igκ was detected in one (1/5) case. Six of the seven cases died and the remaining patient, who was 44-year-old, was alive after 22 months of follow-up.</p><p><b>CONCLUSIONS</b>PCLBCL is rare, predominantly affects elderly male patients. PCLBCL has poor prognosis and high mortality, but younger patients seem to have better prognosis. Some cases had a history of trauma or mosquito bites. The relationship between the history and the onset of PCLBCL needs further evaluation.</p>

Surgical treatment of 43 cases of linear scleroderma of the head and face / 中华皮肤科杂志

Objective To evaluate the performance of surgical approaches in the treatment of linear scleroderma of the head and face. Methods Forty-three patients with stable linear scleroderma of the head and face were included in this study. Surgical approaches, which included direct suture,skin flap translocation, soft-tissue augmentation and filling operation with dermal tissues, were selected and utilized alone or in combination according to the size, location and degree of atrophy and depression of lesions. Results After surgical treatment, wound dehiscence developed in one of these patients, but the wound healed after two sessions of suture. During one year of follow up, all the patients achieved favorite color, feeling and blood supply in the filling areas as well as good facial profile and visual appearance with no obvious convexity and concavity. Skin flaps matched well with surrounding tissues, incision lines were inconspicuous, and hairline looked naturally with well-distributed hairs in these patients after treatment. Patients were satisfied with treatment results. Conclusion Satisfied cosmetic outcomes can be achieved by using surgical approaches in patients with stable linear scleroderma of the head and face.

Application of optical coherence tomography in tissue engineered blood vessel culture based on Luo-Ye pump / 中华胸心血管外科杂志

Objective To explore the application of optical coherence tomography in vascular tissue engineering culture by dynamic monitoring its changes.Methods Human umbilical artery smooth muscle cells were isolated and culture by tissue block method.After passage culture and cell surface markers evaluation, smooth muscle cells were seeded onto polyglycolic acid scaffold and placed into the bioreactor based on Luo-Ye pump with pulsatile stress for three-dimensional culture.At 1、4、 7 、10、14、17、21 days in culture, the image data was obtained by optical coherence tomography technology.The ability of imaging TEBV via OCT was analyzed combined with histopathological observation.Results As the incubation time extended,OCT clearly showed PGA gradual degradation, decreased composite scaffold thickness and the wall structure from loose to tight.At 21 days in culture, the vessel mimics had smooth surface with extracellular matrix evenly distributed and achieved complete reconstruction in the PGA scaffold.Combining with histopathological staining, the blood vessel mimics were similar to natural blood vessels.OCT measured TEBV thickness compared with histopathological measurement had good correlation (r =0.922,P ＜ 0.05).Conclusion Optical coherence tomography could clearly image microstructures of tissue engineered blood vessels cultured in three-dimensional culture system based on Luo-Ye pump, delineate the reconstruction of TEBV-like tissue in the bioreactor and provide as a dynamic and convenient monitoring tool in vascular tissue engineering.

Effects of imatinib mesylate on the biological activity of Hs294T melanoma cells / 中华皮肤科杂志

ObjectiveTo investigate the effects of imatinib mesylate as a tyrosine kinase inhibitor on the biological activity of and Wnt/β-catenin pathway in Hs294T melanoma cells.MethodsAfter Hs294T cells were incubated with imatinib mesylate at various concentrations(4,8,10,16,20 and 24 μmol/L) for 24 hours or imatinib mesylate at 10 μmol/L for 24,48 and 72 hours,methyl thiazolyl tetrazolium (MTT) assay was performed to estimate the proliferation of cells and to determine the effects of imatinib mesylate on the proliferation of Hs294T cells.Then,Hs294T cells were treated with imatinib mesylate at 10 μmol/L or dimethyl sulfoxide (DMSO) for different durations,followed by the detection of cell apoptosis with flow cytometry,localization of β-catenin with annexin V/propidium iodide-double staining and laser confocal microscopy,quantification of β-catenin and cyclin D1 protein with Western blot,and measurement of LEF1 and C-myc mRNA expression with real time fluorescence-based quantitative PCR.Matrigel invasion assay was performed to evaluate the invasiveness of Hs294T cells after treatment with imatinib mesylate at 5 μmol/L or DMSO for 24 hours.ResultsImatinib mesylate at 4-10 μmol/L elicited a dose-dependent decline in the proliferation of Hs294T cells (F =125.3,P ＜ 0.05),and imatinib mesylate at 10 μmol/L induced a time-dependent decrease from 24 to 72 hours(F =714.6,P ＜ 0.01 ).The percentage of early and late apoptotic cells was markedly increased,while the invasiveness was decreased by about 48％(P ＜ 0.01 ),together with a downregulation in the expression of LEF1,C-myc and Cyclin D1 in imatinib mesylate-treated Hs294T cells compared with the DMSO-treated cells.No obvious changes were observed in the protein expression of β-catenin,but a decline in the nuclear localization of β-catenin was noted in Hs294T cells after being treated with imatinib mesylate.ConclusionImatinib mesylate may suppress the proliferation and invasion of,but promote the apoptosis in,melanoma cells,by downregulating the Wnt/β-catenin pathway.

The application of three-dimensional simulated surgical technique in precise hepatectomy / 中华肝胆外科杂志

ObjectiveTo study the application of three-dimensional simulated surgical technique in precise hepatectomy. MethodsFrom July 2009 to February 2010, 16 patients with primary liver cancer underwent preoperative simulated imaging and three-dimensional simulation of liver resection.The 3D extent of simulated hepatectomy and actual hepatectomy was compared and analyzed. ResultsThe shape and the extent of the liver resected were very similar in the simulated and the actual hepatectomies. The mean differences in the length, breadth and depth of the remnant livers were 0. 6118 cm,0. 4490 cm and 0. 3199 cm, respectively. ConclusionsSimulation hepatectomy could predict the extent of the actual liver resection, and provided accurate guidance and preoperative planning for precise hepatectomy.

Resilience and related factors in rescue soldiers / 中华行为医学与脑科学杂志

Objective To research the resilience and related factors of the rescue soldiers. Methods Resilience Scale for Adults( RSA ), Self-report Symptom Checklist 90 (SCL-90), Eysenck Personality Questionnaire(EPQ) and Coping Style Questionnaire were applied in this survey to 1401 rescue soldiers. Results ①The soldiers' resilience score was (2.49±0.57) ,significantly lower than college students( t= 16.619, P=0. 000).The rescue soldiers with the year in army above 3 were significantly higher than that below 2 years in social competence( t=2.581, P=0.01 ) ,social resources( t=2. 158, P=0.031 ) ,structured style( t=3.254, P=0.001 ) and RSA scores(t=2.455, P=0.014). There exist significantly differences in various education level of the rescue soldiers in perception of self(t=3.732, P=0.024), planned of future ( t = 9.324, P = 0.000 ) , social competence ( t = 8. 838, P = 0.000 ), social resources ( t = 13. 660, P = 0. 000 ) and RSA scores (t=9.805, P=0.000) , and college ≥ senior high school ≥ junior high school. ②Compared to the lower group in the RSA, the higher group scored significantly higher in extraversion (t=16. 204, P=0.000) and positive coping( t = - 18. 171, P=0.000), while lower in SCL-90 (t=8.461, P=0.000) and neuroticism (t=8.833, P=0.000 ).③Resilience have the obvious positive correlation with the education ( r= 0. 116 ), extraversion ( r= 0. 463 ) and positive coping(r=0.500) ,and negative correlation with the neuroticism(r=-0.251 ) and SCL-90( r= -0.260). It can be assumed that positive coping,extraversion and neuroticism had good predictive ability(35.9％) to resilience. Conclusion The rescue soldiers have low resilience than college students, positive coping, extraversion, and neuroticism are important psychological factors for rescue soldiers.

Preparation and bioactivity of anti-human red blood cell ScFv and CSFV E2 bifunctional fusion protein / 生物工程学报

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.

Proliferation of and production of interferon-γ by drug-specific peripheral T cells from pafients with severe drug eruption / 中华皮肤科杂志

Objective To detect the proliferation of and production of interferon-γ by drug-specific peripheral T cells from patients with severe drug eruption.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 10 patients with severe drug eruption,10 patients with mild or moderate drug eruption and 10 normal human controls,stimulated with causative drugs to obtain drug-specific T cells.Then,both PBMCs and drug-specific T cells were stimulated with causative drugs or unrelated drugs followed by the detection of secretion levels of IFN-γ with ex vivo enzyme-linked immunodotting (ELISpot) assay and cultured ELlSpot assav respectively.Results After stimulation with causative drugs,a higher level of IFN-γ was secreted by PBMCs and drug-specific T cells from patients with severe drug eruption compared with those from normal human controls (both P<0.01).and by drug-specific T cells than by PBMCs (P<0.01).The culture with unrelated drugs could neither induce the generation of drug-specific T cells nor promote the secretion of IFN-γ by PBMCs from the patients.Drug-specific T cells still existed in the peripheral blood of 3 patients within 1 to 3 years after recovery of drug eruption.Conclusions There are drug-specific T cells in peripheral blood of patients with severe drug eruption,and they may persist for a certain period of time after recovery of drug eruption.Ex vivo ELISpot combined with cultured ELISpot may be applied to the identification of causative drugs in vivo.

Vector construction and expression of soluble mPDL1-hIgGFc and its effect on the proliferation and apoptosis of cells in vitro / 中华微生物学和免疫学杂志

Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.

Construction, Expression and In Vitro Biological Behaviors of Ig scFv Fragment in Patients with Chronic B Cell Leukemia / 华中科技大学学报（医学）（英德文版）

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDSPAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD=0.9921 ku),which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of IdHSP complex vaccine for B-CLL.

Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia / 华中科技大学学报（医学）（英德文版）

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.

The Application of Evidence-based Medicine to the Course of Paediatrics / 中华医学教育探索杂志

Evidence-based medicine(EBM) is a new medical model,which is an inevitable tendency of modern clinical education.This article analyses the advantages and disadvantages of the evidence-based medical education(EBME) and emphasizes the great significance of EBM in paediatrics teaching.

Expression of ?-Catenin and LEF-1 in Malignant Melanoma / 中华皮肤科杂志

Objective To investigate ?-catenin and lymphoid enhancing factor 1 (LEF-1) expression in malignant melanoma. Methods ?-catenin and LEF-1 protein expression was examined using the PowerVisionTM immunohistochemical method in 25 cases of intradermal nevus and 45 cases of malignant melanoma. Results Comparing malignant melanoma with intradermal nevi, there was a significant difference in the expression of ?-catenin (33/45= 73% vs. 9/25 = 36%, respectively; P

Expression and purification of Helicobacter pylori urease A subunit and catalase / 中国免疫学杂志

Objective:Investigated the effect of recombinant urease A subunit and catalase vaccines on protection against Helicobacter pylori (HP) infection Methods:Recombinant plasmids expressing UreA or KatA were constructed Expression was induced with IPTG,analyzed by SDS PAGE and Western blot UreA and KatA were purified with Bulk GST purification module Results:The recombinant plasmids could express GST UreA and GST KatA fusion proteins,which accounted for 35% and 19% of total bacteria proteins respectively,and could specially react with antibody against GST The purity of the purified UreA and KatA was over 95% Conclusion:The work laid a foundation for further studies of HP vaccine The recombinant vaccines may play an important role in preventing and treating HP infection and related diseases

Inhibition of lymphoid enhancer factor-1 expression in human malignant melanoma cell line A375 by RNA interference method / 中华皮肤科杂志

Objective To investigate the inhibition of lymphoid enhancer factor-1 (LEF-1) expression in human malignant melanoma cell line A375 by RNA interference method. Methods Sense and antisense oligonucleotides with hairpin structures, targeted specifically at LEF-1 mRNA, were designed, synthesized, then linked to the expression vector psilencer3.1-H1 neo after annealing. After identification, the re-combinant psilencer3.1-H1/LEF-1 siRNA was used to transfect the cultured A375 cells by a liposome-medi-ated method. The cells expressing the recombinant RNA was detected by G418 screening. The mRNA and protein levels were detected by RT-PCR, Western blotting and immunocytochemistry, respectively. Results The expression vector psilencer3.1-H1/LEF-1 siRNA was successfully constructed, and its stable expression in cell clones was achieved. The mRNA and protein levels of LEF-1 were both down-regulated in the trans-fected cells. Conclusion The recombinant of psilencer3.1-HI/LEF-1 siRNA can inhibit the mRNA and protein expression of LEF-1 in A375 cells.