The colorimetric method has been well developed to detect the sensitivity of most antiproliferative drugs with the advantages of objectivity and accuracy. Many parameters including the dosage and the absorbance of crystalline violet, the extraction time of the solvent, plate effect and marginal effect were investigated, then the optimized method was applied further to measure the biological activity during the refolding of recombinant human IFN-? inclusion bodies.

OBJECTIVETo study the clinicopathological characteristics of synchronous squamous cell carcinoma (SCC) of the renal pelvis and SCC of the ureter.

METHODSThe clinical data of two cases of synchronous SCC of the renal pelvis and SCC of the ureter were retrospectively reviewed and analyzed. In case 1, a 68-year-old man with hematuria for a month, imaging modalities revealed a right renal pelvis tumor and a right distal ureter tumor. The patient underwent nephroureterectomy and excision of the bladder cuff. Case 2, a 60-year-old man with the complaint of lower abdominal pain and left flank pain for a month, was diagnosed as left distal ureteral stone in another hospital. Ureterolithotomy was performed and a ureteral tumor was found at the lower site of the stone intraoperatively. The pathological report demonstrated SCC, and the patient was transferred to our hospital for further treatment. We found a left renal mass invading the left hemicolon during surgery, and nephroureterectomy was performed with a bladder cuff excision, left hemicolon resection, and also complete lymph node dissection. Neither of patients received adjuvant radiotherapy/chemotherapy.

RESULTSModerately differentiated SCC was reported in both of renal pelvis and ureter in case 1 and the tumor invaded the subepithelial connective tissue in the renal pelvis and superficial muscle in the ureter. In case 2, moderately differentiated SCC of the left renal pelvis with colon metastasis and poorly differentiated SCC of the ureter was reported with two retroperitoneal lymph node metastases. The two patients died from tumor recurrence and metastasis 5 and 6 months after the surgery, respectively.

CONCLUSIONSynchronous SCC of the renal pelvis and SCC of the ureter are rare and has high likeliness of early recurrence and metastasis, often with poor prognosis.

Aged ; Carcinoma, Squamous Cell ; complications ; pathology ; Humans ; Kidney Neoplasms ; complications ; pathology ; Kidney Pelvis ; pathology ; Male ; Middle Aged ; Ureteral Neoplasms ; complications ; pathology

AIM:To investigate the value of 3D-optical coherence tomography (OCT) in the diagnosis of macular disease before phacoemulsification.METHODS:Clinical records of 423 cataract patients (512 eyes) who underwent phacoemulsification combined with intraocular lens implantation in our hospital from June to December in 2015 were retrospectively analyzed.In addition to preoperative routine examination of fundus, Topcon 3D-OCT 2000 was used to examine the macula, the detection rate of macular disease was compared, risk factors of cataract combined with macular disease were analyzed.RESULTS:The OCT image results of 305 cases (384 eyes, 72.1%) were successfully obtained, 133 cases showed macular disease (146 eyes), the detection rate was 28.5% (95%CI:27.64%-29.40%);the macular disease of 35 cases (37 eyes) were detected by routine examination of fundus before operation, the detection rate was 7.2% (95%CI:6.72%-7.74%);the detection rate of 3D-OCT was significantly higher than routine examination of fundus for macular disease (χ2=79.05, P<0.01).Female, over 65 years old, surgical history of diseased eye, and high myopia were risk factors of cataract combined with macular disease, the relative risk was 1.705 (95%CI:1.091,2.664), 1.893 (95%CI:1.219,2.939), 6.593 (95%CI:2.027,21.447) and 95%CI:5.130 (2.841,9.263) respectively, the risk of cataract combined with macular disease showed an increasing trend with rising age.CONCLUSION:In preoperative examination of cataract patients, 3D-OCT has higher sensitivity in the detection of macular disease, especially for women, over 65 years old, high myopia and surgical history of diseased eye, 3D-OCT can be used as a routine preoperative examination.

OBJECTIVETo investigate potential mutations and clinical features of 9 unrelated patients with inherited coagulation factor VII (FVII) deficiency.

METHODSClinical diagnosis was validated by assaying of coagulation parameters including prothrombin time, activated partial thromboplastin time, FVII activity and specific antigens. All exons, exon-intron boundaries, and 5' and 3' untranslated regions of F7 genes were amplified with PCR. Potential mutations were detected by direct sequencing of purified PCR products. Suspected mutations were confirmed by sequencing of the opposite strand.

RESULTSAll probands have featured prolonged prothrombin time, with FVII activity ranging between 2.0% to 6.0%. The titers of FVII antigen were significantly reduced in 7 probands. Eight mutations, including 6 missense mutations, 1 deletion and 1 insertion, were identified, among which 3 (Gln100Leu, Ser269Pro and g.11520_11521insT) were not described previously. Six mutations have located in the protease domain. All mutations were inherited, and consanguineous marriages were reported in 5 families. Mutations g.27_28delCT, Cys329Gly, Arg304Trp and His348Gln have been identified in unrelated families. There was a lack of correlation between the mutations and their clinical features. Two individuals with homozygous His348Gln mutations and 1 individual with homozygous Arg304Trp mutation were only mildly affected or asymptomatic. Two patients, who have respectively carried homozygous and heterozygous deletions of g.27_28delCT, were moderately affected and asymptomatic. In 4 patients carrying double heterozygous mutations, 1 (Ser269Pro and Cys329Gly) was asymptomatic, 2 (Arg304Trp and Cys329Gly, Arg277Cys and g.11520_11521insT, respectively) had a mild bleeding tendency, whilst 1 (Gln100Leu and His348Gln) has a moderate bleeding diathesis.

CONCLUSIONThere seem to be hotspots of F7 gene mutations in ethnic Han Chinese populations. And there is a lack of correlation between particular types of mutations and clinical phenotypes.

Adolescent ; Adult ; Aged ; Base Sequence ; Blood Coagulation Disorders, Inherited ; genetics ; Child ; Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Heterozygote ; Homozygote ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Young Adult

OBJECTIVETo achieve high expression of human renal cell carcinoma-associated antigen G250 in Escherichia coli.

METHODSThe gene fragments encoding the protein obtained by PCR was cloned into prokaryotic expression vector pET32a(+) and expressed in E. coli Rosseta. The immunogenicity of the recombinant protein was evaluated by Western blotting.

RESULTSThe plasmid pET32a(+)/G250 was constructed and expressed in E. coli Rosseta successfully. Western blot analysis showed that the recombinant protein could be specifically recognized by monoclonal antibody M75.

CONCLUSIONEfficient G250 expression is achieved in prokaryotic expression system, which may facilitate further functional study of the protein and its monoclonal antibody preparation.

Antibodies, Monoclonal ; immunology ; Antibody Specificity ; immunology ; Antigens, Neoplasm ; genetics ; immunology ; metabolism ; Biomarkers, Tumor ; genetics ; immunology ; metabolism ; Blotting, Western ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

Objective To report a large family with an autosomal dominant dementia associated with mutation in the prion protein gene(PRNP)and the detailed clinical,neuroimaging and pathological manifestations.Methods Two patients from a large family of dementia were admitted to our ward and the data of their medical history,physical examination,video electroenceplialogram,neuroimaging were colleted.A sterotactic biopsy of the right frontal lobe of the proband was done.After the informed consent from the family members obtained,the genomic DNA was extracted from peripheral blood leucocytes of 5 persons followed by in,vitro amplification using polymerase chain reaction(PCR).The PCR products were directly sequenced by Sanger method.PRNP gene sequence was also examined in 150 normal Chinese to exclude single nueleotide polymorphism.Results A missense mutation of PRNP gene in 5 farnily members was detected,resulting in Gll4V mutation in the prion protein,with M/M genotype of eodon 129.This mutation was not detected in 150 normal Chinese.The proband was diagnosed as inherited prion disease by her clinical features,including neuropsychiatrie disturbances and progressive dementia,and manifestations of neuroimaging,EEG,neuropathology and PRNP gene mutation.Conclusion The first autosomal dominant pedigree of family prion disease is found in China with G114V mutation in PRNP gene which may lead to the prion disease directly.

[Summary] A total of 128 individuals with type 2 diabetes underwent continuous glucose monitoring for 3 consecutive days.The dawn phenomenon was defined by three different parameters according to the previous research:(1)the absolute increase of glucose level from nocturnal nadir to prebreakfast value(?G) above 20 mg/dl;(2)?G above 10 mg/dl;( 3 ) insulin requirement increased at least 20%.The participants were secondarily separated by presence/absence of a dawn phenomenon based on the definitions above.The impact on blood glucose fluctuation of different groups was assessed according to the standard deviation of blood glucose( SDBG) , the area under curve above 10 mmol/L ( AUC ) , and the mean amplitude of glycemic excursions ( MAGE ) , etc.The frequencies of dawn phenomenon were 64.8%(?G≥20mg/dl), 85.2%(?G≥10 mg/dl), and 59.4%(rise in insulin requirement≥20%)respectively.The impacts on SDBG, AUC, MAGE, and MODD were without statistical difference(P>0.05) between the presence and absence of the dawn phenomenon patients when?G≥10 mg/dl.However, the differences reached statistical significance(P<0.05) when ?G≥20 mg/dl and the increase in insulin requirement≥20%. Besides, the incidence of dawn phenomenon was positively correlated with HOMA-IR, HbA1C , and free C-peptide.Dawn phenomenon is a very frequent event in type 2 diabetes and not only impacts the overall glycemic control but also exaggerates glucose fluctuation.To be clinically relevant, ?G≥20mg/dl should be taken as the quantitative criterion of the dawn phenomenon.

OBJECTIVETo construct the recombinant adenovirus containing herpes simplex virus-1 virion protein (VP) 22 and human microdystrophin gene, then the adenovirus was transfected into C2C12 myoblast and studied on the property of protein transduction with VP22-mediated microdystrophin in C2C12 myoblast.

METHODSThe full-length VP22 cDNA was obtained from recombinant plasmid pSINrep5-VP22 with PCR, and the product was directionally inserted into pShuttle-CMV to acquire the plasmid pCMV-VP22. Microdystrophin cDNA was obtained from recombinant plasmid pBSK-micro digested with restrictive endonuclease NotI, and the product was directionally inserted into pCMV-VP22 to acquire the plasmid pCMV-VP22-MICDYS. The plasmid of pCMV-VP22-MICDYS was lined with Pme I, and the fragment containing VP22-microdystrophin was reclaimed and transfected into E1 coli BJ5183 with plasmid pAdeasy-1. After having been screened by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the cytopathic effect of cells and by PCR method to acquire the recombinant adenovirus Ad-VP22-MICDYS. Finally, the C2C12 myoblast were transfected with the recombinant adenovirus Ad-VP22-MICDYS and Ad-MICDYS, and the expression of microdystrophin was detected by RT-PCR, Western blot and immunocytochemistry.

RESULTSThe recombinant adenovirus including VP22 and microdystrophin gene was successfully constructed. VP22 transferred VP22-microdystrophin fused protein from infected C2C12 myoblast into uninfected cells and enhance the expression of microdystrophin in myoblast.

CONCLUSIONSRecombinant adenovirus containing VP22 and microdystrophin gene was constructed successfully. VP22 can enhance the expression with microdystrophin in myoblast. It lays the foundation for further studying on VP22-mediated recombinant including microdystrophin gene to cure Duchenne muscular dystrophy.

Adenoviridae ; genetics ; physiology ; Animals ; Cell Line ; Dystrophin ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Mice ; Myoblasts ; metabolism ; virology ; Simplexvirus ; genetics ; metabolism ; Transduction, Genetic ; Viral Structural Proteins ; genetics ; metabolism ; Virion ; genetics ; metabolism

OBJECTIVETo assess the value of fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer.

METHODSUrine samples from 100 patients suspected of having bladder cancer were collected before cystoscopy for immediate urine cytology and FISH analysis. The criteria for FISH abnormality were determined by evaluating the urine specimens from 20 subjects without urogenital neoplasm.

RESULTSThe overall sensitivity of cytology and FISH was 43.2% and 82.4%, and their specificity was 92.3% and 88.5%, with diagnostic concordance rate of 56.0% and 84.0%, respectively. The differences between FISH and cytology showed statistical significance in the sensitivity, diagnostic concordance rate, non-muscle-invasive cancer and primary cancer.

CONCLUSIONThe sensitivity and efficiency of FISH in the detection of bladder cancer are superior to those of cytology, especially for prophase cancer.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Transitional Cell ; diagnosis ; Cytodiagnosis ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Middle Aged ; Sensitivity and Specificity ; Urinary Bladder Neoplasms ; diagnosis ; Urine ; cytology ; Young Adult

OBJECTIVETo investigate the effects of silencing ADP-ribosylation factor 6 (Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC-3 and the possible molecular mechanisms.

METHODSThree Arf6-specific small interfering RNA (siRNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by real-time PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC-3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting.

RESULTSTransfection of siRNA-3 resulted in significantly decreased Arf6 mRNA and protein expression with inhibition rates of (91.88±3.13)% and (86.37±0.57)%, respectively. Arf6 silencing by siRNA-3 markedly suppressed the proliferation, migration and invasion of PC-3 cells and reduced the expression levels of p-ERK1/2 and Rac1.

CONCLUSIONSilencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC-3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.

ADP-Ribosylation Factors ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Humans ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neoplasm Invasiveness ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; Wound Healing ; rac1 GTP-Binding Protein ; metabolism